Sequestration of formaldehyde to stabilize nitrilase specific activity when converting glycolonitrile to glycolic acid

ABSTRACT

A process is provided to improve the specific activity of an enzyme catalyst having nitrilase activity when converting glycolonitrile to glycolic acid under aqueous reaction conditions. Inclusion of an effective amount of at least one amine protectant improves the specific activity and catalytic productivity of the enzyme catalyst.

FIELD OF THE INVENTION

This invention relates to the field of organic acid synthesis, molecularbiology, and microbiology. More specifically, a process is provided toprotect and/or increase the specific activity of an enzyme catalystcomprising a polypeptide having nitrilase activity when convertingglycolonitrile to glycolic acid by conducting the reaction in thepresence of an amine protectant.

BACKGROUND OF THE INVENTION

Glycolic acid (HOCH₂COOH; CAS Registry Number is 79-14-1) is thesimplest member of the α-hydroxy acid family of carboxylic acids. Itsproperties make it ideal for a broad spectrum of consumer and industrialapplications, including use in water well rehabilitation, the leatherindustry, the oil and gas industry, the laundry and textile industry, asa monomer in the preparation of polyglycolic acid (PGA), and as acomponent in personal care products. Glycolic acid also is a principleingredient for cleaners in a variety of industries (dairy and foodprocessing equipment cleaners, household and institutional cleaners,industrial cleaners [for transportation equipment, masonry, printedcircuit boards, stainless steel boiler and process equipment, coolingtower/heat exchangers], and metals processing [for metal pickling,copper brightening, etching, electroplating, electropolishing]).Recently, it has been reported that polyglycolic acid is useful as a gasbarrier material (i.e., exhibits high oxygen barrier characteristics)for packing foods and carbonated drinks (WO 2005/106005 A1). However,traditional chemical synthesis of glycolic acid produces a significantamount of impurities that must be removed prior to use in preparingpolyglycolic acid for gas barrier materials. New technology tocommercially produce glycolic acid, especially one that producesglycolic acid in high purity and at low cost; would be eagerly receivedby industry.

Microbial catalysts can hydrolyze a nitrile (e.g., glycolonitrile)directly to the corresponding carboxylic acids (e.g., glycolic acid)using a nitrilase (EC 3.5.5.7), where there is no intermediateproduction of the corresponding amide (Equation 1), or by a combinationof nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) enzymes,where a nitrile hydratase (NHase) initially converts a nitrile to anamide, and then the amide is subsequently converted by the amidase tothe corresponding carboxylic acid (Equation 2):

Enzymatic synthesis of glycolic acid typically requires a substantiallypure form of glycolonitrile. Methods to synthesize glycolonitrile byreacting aqueous solutions of formaldehyde and hydrogen cyanide havepreviously been reported (U.S. Pat. No. 2,175,805; U.S. Pat. No.2,890,238; and U.S. Pat. No. 5,187,301; Equation 3).

However, these methods typically result in an aqueous glycolonitrilereaction product that requires significant purification (e.g.,distillative purification) as many of the impurities and/or byproductsof the reaction (including excess reactive formaldehyde) may interferewith the enzymatic conversion of glycolonitrile to glycolic acid,including suppression of catalyst activity (i.e., decreased specificactivity). In particular, it is well known that formaldehyde can createundesirable modifications in proteins by reacting with amino groups fromN-terminal amino acid residues and the side chains of arginine,cysteine, histidine, and lysine residues (Metz et al., J. Biol. Chem.,279 (8): 6235-6243 (2004)). Suppression of catalyst activity decreasesthe overall productivity of the catalyst (i.e., total grams of glycolicacid formed per gram of catalyst), adding a significant cost to theoverall process that may make enzymatic production economicallynon-viable when compared to chemical synthesis. As such, reactionconditions are needed that can help to protect the enzymatic activityagainst undesirable impurities reported decrease the activity of thecatalyst.

A method of producing high purity glycolonitrile has been reported bysubjecting the formaldehyde to a heat treatment prior to theglycolonitrile synthesis reaction (U.S. Ser. No. 11/314,386 and U.S.Ser. No. 11/314,905; Equation 3). However, glycolonitrile can reversiblydisassociate into formaldehyde and hydrogen cyanide. As such, thereremains a need to protect a catalyst having nitrilase activity againstthe undesirable effects of free formaldehyde.

Various methods are known for preparing α-hydroxy acids using thecorresponding α-hydroxy nitrite as the starting material and amicroorganism as the catalyst. Examples of α-hydroxy acids producedinclude: glycolic acid, lactic acid, 2-hydroxyisobutyric acid,2-hydroxy-2-phenyl propionic acid, mandelic acid,2-hydroxy-3,3-dimethyl-4-butyrolactone, and 4-methylthiobutyric acid.These products are synthesized using microorganisms, such as thosebelonging to the genera Nocardia, Bacillus, Brevibacterium,Aureobacterium, Pseudomonas, Caseobacter, Alcaligenes, Acinetobacter,Enterobacter, Arthrobacter, Escherichia, Micrococcus, Streptomyces,Flavobacterium, Aeromonas, Mycoplana, Cellulomonas, Erwinia, Candida,Bacteridium, Aspergillus, Penicillium, Cochliobolus, Fusarium,Rhodopseudomonas, Rhodococcus, Corynebacterium, Microbacterium,Obsumbacterium and Gordona, (JP-A4-994951, JP-A-4-99496 andJP-A-4-218385 corresponding to U.S. Pat. No. 5,223,416; JP-A-4-99497corresponding to U.S. Pat. No. 5,234,826; JP-A-5-95795 corresponding toU.S. Pat. No. 5,296,373; JP-A-5-21987; JP-A-5-19189 corresponding toU.S. Pat. No. 5,326,702; JP-A-6-237789 corresponding to EP-A-0610048,JP-A-6-284899 corresponding to EP-A-0610049; JP-A-7-213296 correspondingto U.S. Pat. No. 5,508,181).

However, most known methods for preparing α-hydroxy acids from thecorresponding α-hydroxy nitriles as mentioned above do not produce andaccumulate a product at a sufficiently high concentration to meetcommercial needs. This is frequently a result of enzyme inactivationearly in the reaction period. U.S. Pat. No. 5,756,306 teaches that “Whenan α-hydroxy nitrite is enzymatically hydrolyzed or hydrated usingnitrilase or nitrite hydratase to produce an α-hydroxy acid or α-hydroxyamide, a problem occurs in that the enzyme is inactivated within a shortperiod of time. It is therefore difficult to obtain the α-hydroxy acidor α-hydroxy amide in high concentration and high yield.” (col. 1, lines49-54). Maintaining the aldehyde concentration (formed by thedisassociation of α-hydroxy nitrite to aldehyde and hydrogen cyanide)and/or the α-hydroxy nitrite concentration in the reaction mixturewithin a specified range is one method to avoid this problem.

U.S. Pat. No. 5,508,181 addresses further difficulties relating to rapidenzyme inactivation. Specifically, U.S. Pat. No. 5,508,181 mentions thatα-hydroxy nitrite compounds partially disassociate into thecorresponding aldehydes, according to the disassociation equilibrium.These aldehydes were reported to inactivate the enzyme within a shortperiod of time by binding to the protein, thus making it difficult toobtain α-hydroxy acid or α-hydroxy amide in a high concentration withhigh productivity from α-hydroxy nitriles (col. 2, lines 16-29). As asolution to prevent enzyme inactivation due to accumulation ofaldehydes, phosphate or hypophosphite ions were added to the reactionmixture. U.S. Pat. No. 5,326,702 reports the use of sulfite, disulfite,or dithionite ions to sequester aldehyde and prevent enzymeinactivation, but concludes that the concentration of α-hydroxy acidproduced and accumulated even by using such additives as described aboveis not great.

U.S. Pat. No. 6,037,155 teaches that low accumulation of α-hydroxy acidproduct is related to enzyme inactivation within a short time due to thedisassociated-aldehyde accumulation. These inventors suggest thatenzymatic activity is inhibited in the presence of hydrogen cyanide(Asano et al., Agricultural Biological Chemistry, Vol. 46, pages1165-1174 (1982)) generated in the partial disassociation of theα-hydroxy nitrite in water together with the corresponding aldehyde orketone (Mowry, David T., Chemical Reviews, Vol. 42, pages 189-283(1948)). The inventors solved the problem of aldehyde-induced enzymeinactivation by using microorganisms whose enzyme activity could beimproved by adding a cyanide substance to the reaction mixture. Theaddition of a cyanide substance limited the disassociation of α-hydroxynitrite to aldehyde and hydrogen cyanide.

With specific respect to the production of glycolic acid, glycolonitrileis known to reversibly disassociate to hydrogen cyanide andformaldehyde, either of which may be involved in reducing catalystactivity. U.S. Pat. No. 3,940,316 describes a process for preparing anorganic acid from the corresponding nitrite using bacteria with“nitrilasic” activity, and lists glycolonitrile as a substrate. Inparticular, this patent describes the use of Bacillus, Bacteridium,Micrococcus, and Brevibacterium for this purpose. Though described ashaving nitrilasic activity, Brevibacterium R312 is the only strain usedin all of the U.S. Pat. No. 3,940,316 examples. Brevibacterium R312 isknown to have nitrite hydratase and amidase activities, but no nitrilaseactivity (Tourneix et al., Antonie van Leeuwenhoek, 52:173-182 (1986)).

A method for preparing lactic acid, glycolic acid, and2-hydroxyisobutyric acid by using a microorganism belonging toCorynebacterium spp. is disclosed in Japanese Patent Laid-open No. Sho61-56086. JP 09028390 discloses a method for manufacturing glycolic acidfrom glycolonitrile by the action of Rhodococcus or Gordona hydrolase.Selectivity for glycolic acid is reported as almost 100%, withoutformation of glycolic acid amide. U.S. Pat. No. 6,037,155 disclosesexamples of methods for producing α-hydroxy acids from α-hydroxynitriles, including glycolic acid, This disclosure acknowledges that notall microbial catalysts can produce high concentrations of glycolic aciddue to the aforementioned problems and instructs that screening studiesmust be conducted in order to find industrially advantageousmicroorganisms. U.S. Pat. No. 6,037,155 specifically identifiesVariovorax spp. and Arthrobacter spp. microorganisms that are resistantto the suppressing effect of α-hydroxy nitrile or α-hydroxy acid, havedurable activity, and can produce the desired product at highconcentration.

Acidovorax facilis 72W (ATCC 55746) is characterized by aliphaticnitrilase (EC 3.5.5.7) activity, as well as a combination of nitrilehydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities. The geneencoding the A. facilis 72W (ATCC 55746) nitrilase has been cloned andrecombinantly expressed (WO 01/75077 corresponding to U.S. Pat. No.6,870,038) and Chauhan et al., Appl Microbiol Biotechnol, 61:118-122(2003)).

The A. facilis 72W nitrilase converts α-hydroxynitriles to thecorresponding α-hydroxycarboxylic acids in high yield (U.S. Pat. No.6,383,786), including glycolic acid (U.S. Pat. No. 6,416,980). Animproved process to produce glycolic acid from glycolonitrile usingmutants derived from the A. facilis 72W nitrilase is disclosed inWO2006/068110 and WO2006/069114 (corresponding to U.S. Pat. No.7,198,927 and U.S. patent application Ser. No. 11/314,905,respectively). In co-pending and commonly owned U.S. patent applicationSer. No. 11/314,905, various means to improve production of glycolicacid are disclosed including (1) the use of A. facilis 72W mutantshaving improved activity, (2) the addition of at least one stabilizingagent (e.g. potassium thiosulfate, sodium dithionite, excess HCN), (3)running the reaction under oxygen free conditions, (4) controlling theglycolonitrile feed rate, and (5) the use of high purity glycolonitrile.Even though many of these means improved glycolic acid productivity, adecrease in enzymatic activity was generally observed over time. Thisdecrease in activity is typically attributed, at least in part, to thepresence of formaldehyde (albeit at low levels) in the reaction mixture.A process to protect the specific activity of an enzyme catalyst havingnitrilase activity when converting glycolonitrile to glycolic acid inthe presence of formaldehyde would significantly improve the economicsof glycolic acid synthesis.

The problem to be solved is to provide a process to stabilize and/orincrease the specific activity of an enzyme catalyst having nitrilaseactivity when converting glycolonitrile to glycolic acid in the presenceof formaldehyde.

SUMMARY OF THE INVENTION

The present problem has been solved by providing a process forstabilizing and/or increasing the specific activity of a nitrilasecatalyst comprising a polypeptide having nitrilase activity whenenzymatically converting glycolonitrile to glycolic acid, said processcomprising:

(a) providing a set of reaction components comprising:

-   -   (i) an aqueous solution of glycolonitrile comprising at least        0.01 ppm formaldehyde;    -   (ii) an enzyme catalyst comprising a polypeptide having        nitrilase activity wherein said polypeptide comprises a        catalytic signature motif of SEQ ID NO: 1; wherein said enzyme        catalyst comprises a specific activity for hydrolyzing        glycolonitrile to glycolic acid; and    -   (iii) an effective amount of at least one amine protectant        selected from the group consisting of:        -   a) a compound of the formula

[R₃]_((1+X))—NH_((2−X))

-   -   wherein X is 0 or 1 and R₃ is independently C1 to C20        hydrocarbyl group or substituted hydrocarbyl group; wherein R3        optionally comprises one or more ether linkages; with the        proviso that (i) the substituted group is preferably not a cyano        group capable of reacting with the enzyme catalyst and (ii) R₃        is not a carbonyl group,        -   b) a polyamine polymer comprising an effective number of            free amine groups; and        -   c) an amine-functionalized material comprising an effective            number of free amine groups;    -   wherein the amine protectant is not naturally-produced by said        enzyme catalyst;

(b) combining the set of reaction components under suitable aqueousreaction conditions whereby glycolic acid is produced; wherein saidspecific activity is increased by the addition of said at least oneamine protectant; and

(c) recovering the glycolic acid or salt thereof produced in (b).

BRIEF DESCRIPTION OF THE FIGURE, SEQUENCE LISTING, AND THE BIOLOGICALDEPOSITS

The invention can be more fully understood from the sequence listing,the biological deposits, the FIGURE, and the detailed description thattogether form this application.

FIGURE

FIG. 1, panels A-G, is a CLUSTALW alignment (version 1.83 using defaultparameters) of various nitrilase sequences. The conserved catalystsignature sequence surrounding the catalyst cysteine residue ishighlighted in gray shading. The amino acids representing the catalytictriad (Glu₄₈, Lys₁₃₀, and Cys₁₆₄; numbering based on the amino acidsequence SEQ ID NO: 4) are underlined.

SEQUENCE LISTING

The following sequence descriptions and sequences listings attachedhereto comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Descriptions contain the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IYUB standards describedin Nucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the amino acid sequence of the catalytic signature motifencompassing the essential cysteine residue of nitrilase enzymes(Formula 1).

SEQ ID NO: 2 is the amino acid sequence of a preferred catalystsignature motif encompassing the essential cysteine residue of nitrilaseenzymes (Formula 2)

SEQ ID NO: 3 is the nucleotide sequence of the Acidovorax facilis 72Wnitrilase coding sequence comprising a change in the start codon fromTTG to ATG to facilitate recombinant expression in E. coli.

SEQ ID NO: 4 is the deduced amino acid sequence of the Acidovoraxfacilis 72W nitrilase (ATCC 55746).

SEQ ID NO: 5 is the amino acid sequence of the Alcaligenes faecalis JM3nitrilase (GENBANK® BAA02684.1).

SEQ ID NO. 6 is the amino acid sequence of the Rhodococcus rhodochrousJ1 nitrilase (GENBANK® Q03217).

SEQ ID NO; 7 is the amino acid sequence of the Rhodococcus rhodochrousK22 nitrilase (GENBANK® Q02068).

SEQ ID NO: 8 is the amino acid sequence of the Nocardia sp. C-14-1nitrilase (GENBANK® AAX18182.1).

SEQ ID NO: 9 is the amino acid sequence of the Bordetella bronchisepticaRB50 nitrilase (GENBANK® NP-887662.1).

SEQ ID NO: 10 is the amino acid sequence of the Arabidopsis thaliananitrilase (GENBANK® AAB60275.1 and AMA19627.1).

SEQ ID NO: 11 is the amino acid sequence of the Synechococcus elongatusPCC 7942 nitrilase (GENBANK®YP_(—)399857.1).

SEQ ID NO: 12 is the amino acid sequence of the Synechococcus elongatusPCC 6301 nitrilase (GENBANK®YP_(—)171411.1).

SEQ ID NO: 13 is the amino acid sequence of the Synechocystis sp. PCC6803 nitrilase (GENBANK® NP_(—)442646.1).

SEQ ID NO: 14 is the amino acid sequence of the Pseudomonas entomophilaL48 nitrilase (GENBANK®YP_(—)6090481.1).

SEQ ID NO: 15 is the amino acid sequence of the Zymomonas moblisnitrilase (GENBANK® YP_(—)162942.1).

SEQ ID NO: 16 is the amino acid sequence of the Bacillus sp. OxB-1nitrilase (GENBANK® BAA90460.1).

SEQ ID NO: 17 is the amino acid sequence of the Comamonas testosteroninitrilase (GENBANK®AAA82085.1).

SEQ ID NO: 18 is the amino acid sequence of the Synechococcus sp. CC9605nitrilase (GENBANK® YP_(—)381420.1).

SEQ ID NO: 19 is the amino acid sequence of the Pseudomonas fluorescensPf-5 nitrilase (GENBANK® YP_(—)260015.1).

SEQ ID NO: 20 is the amino acid sequence of the Nocardia farcinica IFM10152 nitrilase (GENBANK®YP_(—)119480.1).

SEQ ID NO: 21 is the amino acid sequence of the Alcaligenes faecalis1650 nitrilase (GENBANK® AAY06506.1).

SEQ ID NO: 22 is the amino acid sequence of the Pseudomonas syringae pv.syringae B728a nitrilase (GENBANK® AAY35081.1).

SEQ ID NO: 23 is the amino acid sequence of the Bradyrhizobium sp. BTAilnitrilase (GENBANK®ZP_(—)00859948.1).

SEQ ID NO: 24 is the amino acid sequence of the Rhodococcus rhodochrousNCIMB 11216 nitrilase (GENBANK® CAC88237).

SEQ ID NO: 25 is the amino acid sequence of Rhodococcus rhodochrousATCC™ 39484

SEQ ID NO: 26 is the nucleotide sequence of an A. facilis 72W nitritasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201Q; Leu

Gln).

SEQ ID NO: 27 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 26) comprising a single amino acid substitution at residueposition 201 (Leu201

Gln) of the A. facilis 72W nitrilase.

SEQ ID NO: 28 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201A; Leu

Ala).

SEQ ID NO: 29 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 28) comprising a single amino acid substitution at residueposition 201 (Leu20

Ala) of the A. facilis 72W nitrilase.

SEQ ID NO: 30 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201C; Leu

Cys).

SEQ ID NO: 31 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 30) comprising a single amino acid substitution at residueposition 201 (Leu201

Cys) of the A. facilis 72W nitrilase.

SEQ ID NO: 32 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201T; Leu

Thr).

SEQ ID NO: 33 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 32) comprising a single amino acid substitution at residueposition 201 (Leu201

Thr) of the A. facilis 72W nitrilase.

SEQ ID NO: 34 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201G; Leu

Gly).

SEQ ID NO: 35 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 34) comprising a single amino acid substitution at residueposition 201 (Leu201

Gly) of the A. facilis 72W nitrilase.

SEQ ID NO: 36 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201H; Leu

His).

SEQ ID NO: 37 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 36) comprising a single amino acid substitution at residueposition 201 (Leu201

His) of the A. facilis 72W nitrilase.

SEQ ID NO: 38 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201K; Leu

Lys).

SEQ ID NO: 39 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 38) comprising a single amino acid substitution at residueposition 201 (Leu201

Lys) of the A. facilis 72W nitrilase.

SEQ ID NO: 40 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201N; Leu

Asn).

SEQ ID NO: 41 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 40) comprising a single amino acid substitution at residueposition 201 (Leu201

Asn) of the A. facilis 72W nitrilase.

SEQ ID NO: 42 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201S; Leu

Ser).

SEQ ID NO: 43 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 42) comprising a single amino acid substitution at residueposition 201 (Leu201

Ser) of the A. facilis 72W nitrilase.

SEQ ID NO: 44 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168K; Phe

Lys).

SEQ ID NO: 45 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 44) comprising a single amino acid substitution at residueposition 168 (Phe168

Lys) of the A. facilis 72W nitrilase.

SEQ ID NO: 46 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168M; Phe

Met).

SEQ ID NO: 47 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO. 46) comprising a single amino acid substitution at residueposition 168 (Phe168

Met) of the A. facilis 72W nitrilase.

SEQ ID NO: 48 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168T; Phe

Thr).

SEQ ID NO: 49 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 48) comprising a single amino acid substitution at residueposition 168 (Phe168

Thr) of the A. facilis 72W nitrilase.

SEQ ID NO: 50 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168V; Phe

Val).

SEQ ID NO: 51 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO:50) comprising a single amino acid substitution at residueposition 168 (Phe168

Val) of the A. facilis 72W nitrilase.

SEQ ID NO: 52 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (T210A; Thr

Ala).

SEQ ID NO: 53 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 52) comprising a single amino acid substitution at residueposition 210 (Thr210

Ala) of the A. facilis 72W nitrilase.

SEQ ID NO: 54 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (T210C; Thr

Cys).

SEQ ID NO, 55 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 54) comprising a single amino acid substitution at residueposition 210 (Thr210

Cys) of the A. facilis 72W nitrilase.

SEQ ID NO: 56 is the nucleotide sequence of the A. facilis 72W nitrilaseexpressed in E. coli strain SS1001 (ATCC PTA-1177).

SEQ ID NO: 57 is the deduced amino acid sequence of the mutant A.facilis 72W nitrilase expressed in E. coli SS1001 (ATCC PTA-1177).

BIOLOGICAL DEPOSITS

The following biological deposits have been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure:

Depositor Identification Int'l. Depository Reference Designation Date ofDeposit Acidovorax facilis 72W ATCC 55746 8 Mar. 1996 E. coli SS1001ATCC PTA-1177 11 Jan. 2000

As used herein, “ATCC” refers to the American Type Culture CollectionInternational Depository Authority located at ATCC, 10801 UniversityBlvd., Manassas, Va. 20110-2209, USA. The “International DepositoryDesignation” is the accession number to the culture on deposit withATCC.

The listed deposits will be maintained in the indicated internationaldepository for at least thirty (30) years and will be made available tothe public upon the grant of a patent disclosing it. The availability ofa deposit does not constitute a license to practice the subjectinvention in derogation of patent rights granted by government action.

DETAILED DESCRIPTION OF THE INVENTION

A process is provided to stabilize and/or increase the specific activityof an enzyme catalyst having nitrilase activity when convertingglycolonitrile to glycolic acid under suitable aqueous reactionconditions, wherein the reaction conditions includes an effective amountof at least one amine protectant.

DEFINITIONS

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like.

The term “about” also encompasses amounts that differ due to differentequilibrium conditions for a composition resulting from a particularinitial mixture. Whether or not modified by the term “about”, the claimsinclude equivalents to the quantities. In one embodiment, the term“about” means within 10% of the reported numerical value, preferablywithin 5% of the reported numerical value.

As used herein, the terms “amine protectant”, “exogenously added amineprotectant”, and “protectant” are used interchangeably to refer to acompound included in the reaction mixture that is notnaturally-produced, isolated, or a naturally a portion of the nitrilasecatalyst, wherein the amine protectant comprises an effective amount ofat least one primary amine group (R—NH₂) and/or secondary amine group(R—NH—R₂) wherein R and R₁ may be the same or different and whereinneither R or R₂ are carbonyl groups (i.e., the primary or secondaryamine group is not part of an amide group). In a preferred embodiment,the amine protectant comprises an effective number of primary aminegroups. In one embodiment, the amine protectant is elected from thegroup consisting of polyamine polymers and amine-functionalizedmaterials including amine-functionalized support materials andamine-functionalized polymers.

As used herein, the term “effective amount of an amine protectant” or“effective amount of at least one amine protectant” will refer to anamount of the amine protectant that improves the nitrilase catalyststability, specific activity, and/or catalytic productivity whenconverting glycolonitrile to glycolic acid (or the ammonium saltthereof. In one embodiment, the “effective amount of amine protectant”refers to the amount of amine protectant present in the reaction mixturethat results in a measured increase in the catalyst's specific activitywhen compared to the specific activity of the same catalyst in theabsence of the amine protectant under identical reaction conditions. Itis known that an aqueous solution of glycolonitrile typically containsfree formaldehyde and formaldehyde-derived impurities produced duringthe synthesis of glycolonitrile or generated as the result ofglycolonitrile dissociation. It is understood that the amine protectantwill have an “effective amount of free primary and/or secondary aminegroups” capable of reacting with the formaldehyde orformaldehyde-derived impurities. By proviso, the amine protectant doesnot include compounds naturally produced by or naturally present in thenitrilase catalyst (for example, natural compounds produced by or withina whole cell catalyst or those found within a whole cell extract).

As used herein, the term “amine-functionalized” or “amine functionalizedmaterial” refers to a compound or material that has been chemicallymodified to have one or more primary and/or secondary amine groups. Inone embodiment, the amine-functionalized material is selected from thegroup consisting of amine-functionalized support materials andamine-functionalized polymers. Means to chemically-functionalizecompounds to contain one or more amine groups are well-known in the art(see, for example, Bickerstaff, G. F., Immobilization of Enzymes andCells, Methods in Biotechnology, Volume 1. 1997. Humana Press, Totowa,N. J., and Wong, S. S., Chemistry of Protein Conjugation andCrosslinking, 1991. CRC Press, Boca Raton, Fla.).

As used herein, “amine-functionalized support materials” include, butare not limited to inorganic materials such as controlled pore glass andglass beads (e.g. aminopropylsilated glass beads), silica, magnetite,and alumina as well as support materials often used weakly basic anionexchange resins as well as ω-aminohexylagarose, ω-aminododecylagarose,and ω-aminoethylagarose. In one embodiment, the amine-functionalizedmaterial comprises polyethylenimine (i.e. “apolyethylenimine-functionalized material”).

As used herein, the term “amine-functionalized polymers” refers topolymers that have been modified to include one or more primary and/orsecondary amine groups. Typically, the non-functionalized polymer doesnot normally contain an effective number of free amine groups.

As used herein, the terms “free amine group” refers to a primary aminegroup (R—NH₂) and/or a secondary amine group (R—NH—R₂) on the amineprotectant that is capable of reacting with formaldehyde.

As used herein, the term “polyamine polymers” refers to polymerscomprising one or more free amine groups. Polyamine polymers areprepared from at least one monomer comprising at least one primaryand/or at least one secondary amine group. In one embodiment, thepolyamine polymers include copolymers comprising at least one free aminegroup. Polyamine polymers may range in size from oligomers (typicallyless than 1,000 Daltons) to higher molecule weight polymers ranging from1,000 Daltons to 10,000,000 Daltons, preferably from 1,000 Daltons to2,000,000 Daltons, more preferably 1,000 Daltons to 1,000,000 Daltons,and more preferably from 10,000 Daltons to 1,000,000 Daltons.

As used herein, the term “glycolonitrile” is abbreviated as “GLN” and issynonymous with hydroxyacetonitrile, 2-hydroxyacetonitrile,hydroxymethylnitrile, and all other synonyms of CAS Registry Number107-164.

As used herein, the term “glycolic acid” is abbreviated as ‘GLA’ and issynonymous with hydroxyacetic acid, hydroxyethanoic acid, and all othersynonyms of CAS Registry Number 79-14-1. The glycolic acid produced bythe present processes may in the form of the protonated carboxylic acidand/or the corresponding ammonium salt.

As used herein, the term ‘ammonium glycolate’ is abbreviated “NH₄GLA”.

As used herein, the term “glycolamide” is the amide derived from thereaction of ammonia with glycolic acid and refers to all other synonymsof compounds having CAS Registry Number 598-42-5.

As used herein, the term “glycolide” refers to the compound of CASRegistry Number 502-97-6.

As used herein, the term “formaldehyde” is abbreviated as “FA” and issynonymous with formic aldehyde, methyl aldehyde, oxomethane, and allother synonyms of CAS Registry Number 50-00-0. Commercially availableformaldehyde is typically comprised of a mixture of monomericformaldehyde (“free formaldehyde”) and various oligomers of formaldehydealong with some methanol (typically about 1 wt % to about 15 wt %).

As used herein, the term “hydrogen cyanide” is synonymous with prussicacid, hydrocyanic acid, and all other synonyms of CAS Registry Number200-821-6.

As used herein, the term “recovering” means isolating, purifying, ortransferring the product formed by the present process. Methods toisolate and purify the product(s) from the reaction mixture are wellknown in the art may include, but are not limited to selectiveprecipitation, crystallization, filtration, reactive solvent extraction,ion exchange, electrodialysis, polymerization, distillation, thermaldecomposition, alcoholysis, column chromatography, and combinationsthereof. In one embodiment, the term “recovering” may also includetransferring the product mixture (typically after filtering out theenzyme catalyst) to another reaction to create one or more additionalproducts. In a preferred embodiment, ion exchange is used to recover theglycolic acid.

As used herein, the terms “enzyme catalyst”, “nitrilase catalyst” or“microbial cell catalyst” refers to a catalyst that is characterized bya nitrilase activity (i.e., comprises at least one polypeptide havingnitrilase activity) for converting glycolonitrile to glycolic acid andammonia. A nitrilase enzyme directly converts a nitrile (preferably, analiphatic nitrile) to the corresponding carboxylic acid, without formingthe corresponding amide as intermediate (see Equation 1). Nitrilasesshare several conserved signature domains known in the art including asignature domain herein referred to as the “catalytic signaturesequence” or “signature sequence”. This region comprises an essentialcysteine residue (e.g., Cys₁₆₄ of SEQ ID NO: 4). As such, polypeptideshaving nitrilase activity can be identified by the existence of thecatalytic domain signature sequence (SEQ ID NO: 1). In a preferredembodiment, the signature sequence is SEQ ID NO: 2. The enzyme catalystmay be in the form of a whole microbial cell, permeabilized microbialcell(s), one or more cell components of a microbial cell extract,partially purified enzyme, or purified enzyme. The enzyme catalyst canbe free (unimmobilized) or immobilized in or on a soluble or insolublesupport. As used herein, “recycled enzyme catalyst” refers to an enzymecatalyst that is reused as an enzyme catalyst in batch reactions. In apreferred embodiment, the enzyme catalyst is a transformed microbialhost cell recombinantly expressing at least one enzyme having nitrilaseactivity; wherein said enzyme comprises the catalytic signature motif ofSEQ ID NO: 1 or SEQ ID NO: 2.

As used herein, the terms “Acidovorax facilis” and “A. facilis” are usedinterchangeably and refer to Acidovorax facilis 72W deposited to theAmerican Type Culture Collection (an international depository authority)having accession number 55746 (“ATCC 55746”). The mutant nitrilasesderived from A. facilis 72W characterized by improved nitrilase activitywhen converting glycolonitrile to glycolic acid have been previouslyreported (see co-owned U.S. Pat. No. 7,198,927). Examples of these A.facilis 72W-derived mutant nitrilases are provide by SEQ ID NOs: 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55.

As used herein, the terms “Escherichia coli” and “E. coli” are usedinterchangeably. Several strains of E. coli suitable for recombinantexpression are described herein including, but not limited to E. coliMG1655 having international depository number ATCC 47076, E. coli FM5having international depository number ATCC 53911, E. Coli W3110 havinginternational depository number ATCC 27325, E. coli MC4100 havinginternational depository number ATCC 35695, and E. coli W1485 havinginternational depository number ATCC 12435, In one embodiment, suitableEscherichia coli strains include E. coli FM5 (ATCC 53911) and E. coliMG1655 (ATCC 47076).

As used herein, the terms “E. coli SS1001” or “SS1001” refer to atransformed E. coli strain expressing the Acidovorax facilis 72Wnitrlase having ATCC Accession No. PTA-1177 (see U.S. Pat. No.6,870,038; herein incorporated in its entirety by reference). Therecombinantly expressed E. coli SS81001 nitrilase (SEQ ID NO: 57)contains 2 minor sequence changes in comparison to the wild-type 72Wnitrilase sequence (SEQ ID NO: 4). The start codon was changed from GTGto ATG to facilitate recombinant expression and an artifact wasintroduced during cloning that resulted in a single amino acid changenear the C-terminal (Pro367 [CCA]

Ser [TCA]).

As used herein, the terms “suitable aqueous glycolonitrile reactionmixture”, “suitable aqueous reaction mixture”, and “suitable reactionconditions” refer to the materials (including at least one amineprotectant) and reaction conditions under which the present process isconducted. The components of the suitable aqueous reaction mixture areprovided herein and those skilled in the art appreciate the range ofcomponent variations suitable for this process.

As used herein, the terms “aqueous ammonium glycolate solution”,“aqueous solution comprising ammonium glycolate”, and “aqueous solutionof ammonium glycolate” will be used to describe an aqueous solutioncomprising ammonium glycolate produced by the enzymatic hydrolysis ofglycolonitrile under typical enzymatic reaction conditions (i.e., a pHrange of about 6 to about 8). The aqueous solution of ammonium glycolatecomprises ammonium glycolate at a concentration of at least about 0.1weight percent (wt %) to about 99 wt % ammonium glycolate. In anotherembodiment, the aqueous solution of ammonium glycolate is comprised ofat least about 10 wt % to about 75 wt % ammonium glycolate. In a furtherembodiment, the aqueous solution of ammonium glycolate is comprised ofat least about 20 wt % to about 50 wt % ammonium glycolate. The pH ofthe aqueous solution of ammonium glycolate can be about 2 to about 12,preferably 5 to about 10, more preferably 6 to about 8. The pH may beadjusted as needed prior to initiating process steps related torecovering glycolic acid (in the form of the acid or salt) from theaqueous ammonium glycolate solution.

As used herein, the terms “catalyst productivity” and “enzyme catalystproductivity” refer to the total amount of product produced per gram ofcatalyst. In the present process, the catalyst comprises a nitrilaseenzyme (EC 3.5.5.7) and the product formed is glycolic acid and/orammonium glycolate (depending upon the pH of the reaction). In general,the present methods are conducted under essentially pH neutralconditions so that the glycolic acid produced is predominantly in theform of the corresponding salt of glycolic acid (i.e. ammoniumglycolate). Generally, in batch reactions with catalyst recycle, thecatalyst activity decreases with each recycle reaction (enzymeinactivation). As shown in Example 6, and in one embodiment, thespecific activity of the catalyst is increased over multiple recyclereactions, resulting in an increase in catalyst productivity.

The terms “catalyst specific activity”, “specific activity”, and“nitrilase specific activity” refers to the enzyme activity per unitmass (for example, milligram) of protein, dry cell weight, or beadweight (immobilized catalyst) when converting glycolonitrile to glycolicacid (or the corresponding ammonium glycolate). Comparisons in nitrilaseactivity were measured proportional to the dry cell weight or beadweight. Since the same catalyst and expression system was used tocompare the specific activity between reactions with or without (i.e.the control) the amine protectant, comparisons and reportedimprovements/increases in nitrilase specific activity were measuredrelative to dry cell weight (dcw) or bead weight (bw). As used herein,the term “improved catalyst specific activity”, and “improved specificactivity” refers to the increase in specific activity observed relativeto the specific activity of the corresponding control. The improvementin specific activity is measured under identical reaction conditionswherein the only different is the inclusion or omission of the amineprotectant. In one embodiment, the improvement in specific activity ismeasured over multiple recycle reactions. In another embodiment, theimprovement or increase in catalyst specific activity using the presentprocess is increased at least 1%, preferably at least 5%, morepreferably at least 10%, even more preferably at least 25%.

As used herein, the term “one unit of enzyme activity” or “one unit ofnitrilase activity” or “U” is defined as the amount of enzyme activityrequired for the production of 1 μmol of glycolic acid product perminute (GLA U/g dry cell weight or bead weight) at a specifiedtemperature (e.g. 25° C.).

As used herein, the terms “recombinant organism”, “transformed hostcell”, “host cell”, “transformant”, “transgenic organism”, and“transformed microbial host” refer to a host organism having beentransformed with heterologous or foreign DNA. The recombinant organismsof the present invention express foreign coding sequences or genes thatencode active nitrilase enzyme. “Transformation” refers to the transferof a DNA fragment into the host organism. The transferred DNA fragmentcan be chromosomally or extrachromosomally incorporated (i.e., via avector) into the host organism. As used herein, the term “transformationcassette” refers to a specific fragment of DNA containing a set ofgenetic elements conveniently arranged for insertion into a host cell,usually as part of a plasmid. As used herein, the term “expressioncassette” refers to a specific fragment of DNA containing a set ofgenetic elements conveniently arranged for insertion into a host cell,usually as part of a plasmid, which also allows for enhanced geneexpression in the host.

As used herein, the terms “nucleic acid fragment” and “nucleic acidmolecule” refer to DNA molecule that may encode an entire gene, codingsequence, and/or regulatory sequences preceding (5′, upstream) orfollowing (3′, downstream) the coding sequence. In one aspect, thepresent nucleic acid molecules encode for polypeptides having nitrilaseactivity.

As used herein, the term “gene” refers to a nucleic acid molecule thatexpresses a specific protein. As used herein, it may or may notincluding regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. ‘Chimeric gene’refers to any gene that is not a native gene, comprising regulatory andcoding sequences that are not found together in nature. Accordingly, achimeric gene may comprise regulatory sequences and coding sequencesthat are derived from different sources, or regulatory sequences andcoding sequences derived from the same source, but arranged in a mannerdifferent than that found in nature. “Endogenous gene” refers to anative gene in its natural location in the genome of an organism. A“foreign” gene refers to a gene not normally found in the host organism,but that is introduced into the host organism by gene transfer. Foreigngenes can comprise native genes inserted into a non-native organism, orchimeric genes. A “transgene” is a gene that has been introduced intothe genome by a transformation procedure.

As used herein, the term “coding sequence” refers to a DNA sequence thatcodes for a specific amino acid sequence. As used herein, ‘suitableregulatory sequences’ refer to nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing sites, effector binding sites, and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. Promoters that cause a gene to beexpressed in most cell types at most times or under most environmentalconditions are commonly referred to as “constitutive promoters”.Promoters that cause a gene to be expressed only in the presence of aparticular compound or environmental condition are commonly referred toas “inducible promoters”. Since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid molecule so that thefunction of one sequence is affected by the other. For example, apromoter is operably linked with a coding sequence when it is capable ofaffecting the expression of that coding sequence (i.e., that the codingsequence is under the transcriptional control of the promoter). Codingsequences can be operably linked to regulatory sequences in sense orantisense orientation.

As used herein, the term “3′ non-coding sequences” refers to DNAsequences located downstream of a coding sequence and includepolyadenylation recognition sequences (normally limited to eukaryotes)and other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal (normallylimited to eukaryotes) is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor.

The skilled artisan is well aware of the “codon-bias” exhibited by aspecific host cell in using nucleotide codons to specify a given aminoacid. Therefore, when synthesizing a gene for improved expression in ahost cell, it is desirable to design the gene such that its codon usagereflects the preferred codon bias of the host cell. A survey of genesderived from the host cell where sequence information is available candetermine its codon bias. Codon-optimization is well known in the artand has been described for various systems including, but not limited toyeast (Outchkourov et al, Protein Expr Purif, 24(1):18-24 (2002)) and E.coli (Feng et al., Biochemistry, 39(50):15399-15409 (2000)).

Nitrilase Catalysts

All nitrilases (EC 3.5.5.7) share a conserved catalytic triad (Glu, Lys,and Cys) (Chauhan et al., Appl. Microbiol. Biotechnol. 61:118-122(2003); Pace, H. and Brenner, C., Genome Biol. 2(1):reviews0001.1-0001.9 (2001)). All known nitrilases have a nucleophilic cysteinein the enzyme active site (Cowan et al., Extremophiles, 2.207-216(1998); Pace, H. and Brenner, C., supra; and Chauhan et al, supra) andall are susceptible to inactivation by thiol reagents (1.0 mMconcentrations of copper chloride, silver nitrate, mercuric acetate, orferric chloride each produced major decreases in A. facilis 72Wnitrilase enzyme activity). Cysteine residues are also capable of beingirreversibly oxidized to sulfinic acids, resulting in a loss of enzymeactivity. Despite the sensitivity of nitrilase enzymes to variousinactivating mechanisms, immobilized A. facilis 72W cells are robust,capable of retaining much of their nitrilase activity after numerousrecycle reactions (U.S. Pat. No. 6,870,038; U.S. Pat. No. 7,148,051;U.S. Pat. No. 7,198,927; and Chauhan et al., supra). Nitrilase catalystsderived from the A. facilis 72W nitrilase also been shown to catalyzethe conversion of α-hydroxynitriles (i.e., glycolonitrile) toα-hydroxycarboxylic acids (i.e., glycolic acid) (see U.S. Pat. No.6,383,786; U.S. Pat. No. 6,416,980; and U.S. Pat. No. 7,198,927).

Sequence comparisons of the A. facilis 72W nitrilase to other bacterialnitrilases have been reported (U.S. Pat. No. 6,870,038; Chauhan et al.,supra). The 72W nitrilase has several conserved signature domainsincluding a 16-amino acid region near the amino terminus (amino acidresidues 40-55 of SEQ ID NO: 4) and a 12 amino acid catalytic region(amino acid residues 160-171 of SEQ ID NO: 4) containing the essentialcysteine residue. This essential cysteine residue (Cys₁₆₄ of SEQ ID NO:4), along with conserved glutamic acid (Glu₄₈ of SEQ ID NO:4) and lysineresidues (Lys₁₃₀ of SEQ ID NO:4), form the catalytic triad motif foundin all nitrilases (Pace, H., and Brenner, C., supra).

The regions surrounding each of the catalytic triad residues are highlyconserved, especially the region surrounding the catalytic cysteineresidue. The essential catalytic cysteine residue is located with ahighly conserved region referred to as the “catalytic signature motif”or “signature motif”. As such, the present process is useful forprotecting the enzymatic activity of any nitrilase comprising thecatalytic signature motif defined by Formula 1 (bold indicates strictlyconserved amino acid residues, italicized residues are those thatexhibit minimal variability [i.e., minimal variation of 3 or fewer aminoacid residues], the catalytic cysteine residue is underlined):

Formula 1 (SEQ ID NO: 1) Gly-Xaa ₁-Xaa ₂-Xaa₃- Cys -Trp-Glu-Xaa₄-Xaa₅-Xaa₆- Xaa ₇-Xaa ₈.wherein

Xaa₁=Ala or Gly;

Xaa₂=Leu, Val, or Ala;

Xaa₃=Ala, Asn, Ile, Cys, Val, or Gln;

Xaa₄=His or Asn;

Xaa₅=Leu, Tyr, Phe, Ala, Met, Lys, Val, Thr, or Arg;

Xaa₆=Asn, Gln, Met, Leu, or Ser;

Xaa₇=Pro or Thr; and

Xaa₈=Leu or Val.

In a preferred embodiment, the nitrilase signature motif of Formula 1 isXaa₁=Ala or Gly; Xaa₂=Leu; Xaa₃=Ala, Asn, Ile, Cys, Val, or Gln;Xaa₄=His; Xaa₄=Leu, Tyr, Phe, Ala, Met, Lys, Val, Thr or Arg; Xaa₆=Ser,Gln, Asn, or Met; Xaa₇=Pro; and Xaa₈=Leu; resulting in the catalyticsignature motif represented by the following:

(SEQ ID NO: 2) Gly-Xaa₁-Leu-Xaa₃-Cys-Trp-Glu-His-Xaa₅-Xaa₆-Pro- Leu

Examples of nitrilases, including the sequences and position of thecorresponding catalytic signature motif sequence, are provided in Table1.

TABLE 1 Conserved Catalytic Cysteine Region- Catalytic Signature MotifsSequence of Signature Nitrilase GenBank ® Acces- Amino Acid Motif (aminoacid Source sion Number SEQ ID NO. residue positions) Acidovorax FacilisABD98457.1 4 GGLNCWEHFQPL 72W (160-171) Alcaligenes BAA02684.l 5GALCCWEHLSPL faecalis JM3 (159-170) Rhodococcus Q03217 6 GALNCWEHFQTLrhodochrous J1 (161-172) Rhodococcus Q02068 7 GGLNCWEHFQPL rhodochrousK22 (166-177) Nocardia sp. AAX18182.1 8 GGLNCWEHFQPL C-14-1 (154-165)Bordetella NP_867662.1 9 GAVVCWENYMPL bronchiseptica RB50 (161-172)Arabidopsis AAB60275.1 10 GAAICWENRMPL thaliana AAA19627.1 (175-186)Synechococcus YP_399857.1 11 GALACWEHYNPL elongatus PCC 7942 (157-168)Synechococcus YP_171411.1 12 GALACWEHYNPL elongatus PCC 6301 (157-168)Synechocystis sp. NP_442646.1 13 GALACWEHYNPL PCC 6803 (165-176)Pseudomonas YP_6090481.1 14 GAAVCWENYMPL entomophila L48 (161-172)Zymomonas moblis YP_162942.1 15 GAAICWENYMPV (161-172) Bacillus sp.OxB-1 BAA90460.1 16 GGLQCWEHFLPL (158-169) Comamonas AAA82085.1 17GGLQCWEHALPL testosteroni (159-170) Synechococcus sp. YP_381420.1 18GALACWEHYNPL CC9605 (156-167) Pseudomonas YP_260015.1 19 GAVICWENMMPLfluorescens Pf-5 (161-172) Nocardia farcinica YP_119480.1 20GALCCWEHLQPL IFM 10152 (159-170) Alcaligenes AAY06506.1 21 GALCCWEHLSPLfaecalis 1650 (159-170) Pseudomonas AAY35081.1 22 GALCCWEHLQPL syringaepv. (157-168) syringae B728a Bradyrhizobium sp. ZP_00859948.1 23GALCCWEHLQPL BTAiI (163-174) Rhodococcus CAC88237 24 GALNCWEHFQTLrhodochrous (161-172) NCIMB 11216 Rhodococcus N/A 25 GALNCWEHFQTLrhodochrous ATCC (161-172) 30494 ™

In one embodiment, the nitrilase catalyst comprises a polypeptide havingnitrilase activity isolated from a genera selected from the groupconsisting of Acidovorax, Rhodococcus, Nocardia, Bacillus, andAlcaligenes. In one embodiment, the nitrilase catalyst comprises apolypeptide having nitrilase activity isolated from a genera selectedfrom the group consisting of Acidovorax and Rhodococcus.

In another embodiment, the polypeptide having nitrilase activity isderived from Acidovorax facilis 72W (ATCC 55746) or a polypeptide(having nitrilase activity) that is substantially similar to theAcidovorax facilis 72W nitrilase (SEQ ID NO: 4) or the A. facilis 72Wderived enzyme represented by SEQ ID NO: 51.

In one embodiment, the nitrilase catalyst is a microbial host celltransformed to express at least one polypeptide having nitrilaseactivity. In one embodiment the transformed host cell is selected fromthe group consisting of Comamonas sp., Corynebacterium sp.,Brevibacterium sp., Rhodococcus sp., Azotobacter sp., Citrobacter sp.,Enterobacter sp., Clostridium sp., Klebsiella sp., Salmonella sp.,Lactobacillus sp., Aspergillus sp., Saccharomyces sp., Yarrowia sp.,Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp., Candida sp.,Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp., Torulopsissp., Methylobactetia sp., Bacillus sp., Eschenchia sp., Pseudomonas sp.,Rhizobium sp., and Streptomyces sp. In a preferred embodiment, themicrobial host cell is selected from the group consisting of Bacillussp., Pseudomonas sp., and Escherichia sp. In a preferred embodiment, thecatalyst is an Escherichia coli host cell recombinantly expressing oneor more of the polypeptides having nitrilase activity.

In another embodiment, the nitrilase catalyst comprises a polypeptidehaving nitrilase activity wherein said polypeptide having nitrilaseactivity has at least 60% identity to SEQ ID NO: 51, preferably at least70% identity to SEQ ID NO: 51, even more preferably at least 80%identity to SEQ ID NO: 51, yet even more preferably at least 90%identity to SEQ ID NO: 51, and most preferably at least 95% identity toSEQ ID NO: 51.

Working examples of several catalysts having nitrilase activity derivedfrom various sources are described herein, including a catalyst derivedfrom the A. facilis 72W nitrilase. Various mutants derived from theAcidovorax facilis 72W nitrilase enzyme have been reported in the art(U.S. Pat. No. 7,148,051 and U.S. Pat. No. 7,198,927).

In one embodiment, the polypeptide having nitrilase activity is selectedfrom the group consisting of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57. In another embodiment,the polypeptide having nitrlase activity is selected from the groupconsisting of 4, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, and 57. In another embodiment, the polypeptide havingnitrilase activity is selected from the group consisting of 4, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57. In anotherembodiment, the polypeptide having nitrilase activity is selected fromthe group consisting of 4, 24, 25, and 51. In another embodiment, thenitrilase catalyst comprises the polypeptide of SEQ ID NO: 51.

Amine Protectants

The amine protectant is any compound exogenously added to the aqueousreaction mixture (e.g., not naturally produced by, naturally found with,or naturally isolated from the enzyme catalyst) comprising at least oneprimary amine group (R—NH₂) and/or secondary amine group (R—NH—R₂)capable of reacting with formaldehyde wherein R and R₁ may be the sameor different and wherein neither R or R₂ are carbonyl groups (i.e. notan amide group). It is also understood that the present enzyme catalysthydrolyzes nitrite groups, and thus, the amine protectant preferablydoes not include a nitrile group capable of being hydrolyzed by anitrilase catalyst. In a preferred embodiment, the amine protectant isan exogenously added compound or component in the reaction mixturecomprising at least one primary amine group.

In one embodiment of the present invention, an effective amount of atleast one amine protectant is added to the reaction mixture. As usedherein, “an effective amount of an amine protectant” is the amount ofamine protectant necessary to result in a detectable improvement incatalyst specific activity, stability, or catalytic productivity whenenzymatically converting glycolonitrile to glycolic acid. It is wellwithin the skill of one in the art to adjust the amount of amineprotectant in the aqueous reaction mixture in order to achieve thedesired effect. In one aspect, the amount of amine protectant added tothe reaction mixture comprises a molar excess of free amine groupsrelative to the amount of formaldehyde present in the reaction mixture.In one aspect, the reaction mixture comprises at least 0.01 ppmformaldehyde. In another aspect, the amount of amine protectant in thereaction mixture comprises a molar ratio of free amine groups (primary,secondary, or a combination thereof) relative to the amount of freeformaldehyde (HCHO) present in the reaction mixture ranging from0.001:1.0 to 1.0:0.001, preferably at least 0.5:1.0.

In one aspect, the amine protectant is selected from the groupconsisting of:

-   -   a) a compound of the formula

[R₃]_((1+X))—NH_((2−X))

wherein X is 0 or 1 and R₃ is independently C1 to C20 hydrocarbyl groupor substituted hydrocarbyl group, wherein R₃ is optionally comprisingone or more ether linkages with the proviso that (i) the substitutedgroup is preferably not a cyano group capable of reacting with thenitrilase catalyst, and (ii) R₃ is not a carbonyl group,

-   -   b) a polyamine polymer comprising an effective number of free        amine groups; and    -   c) an amine-functionalized material comprising an effective        number of free amine groups;

wherein the amine protectant is not naturally-produced by the enzymecatalyst.

The hydrocarbyl group of R₃ can be linear, branched, cyclic, polycyclic,or aromatic. As used herein, the terms “hydrocarbyl”, “hydrocarbylgroup”, and “hydrocarbyl moiety” is meant a straight chain, branched orcyclic arrangement of carbon atoms connected by single, double, ortriple carbon to carbon bonds and/or by ether linkages, and substitutedaccordingly with hydrogen atoms. Such hydrocarbyl groups may bealiphatic and/or aromatic. Examples of hydrocarbyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl,cyclobutyl, pentyl, cyclopentyl, methylcyclopentyl, hexyl, cyclohexyl,benzyl, and phenyl. In a preferred embodiment, the hydrocarbyl moiety isa straight chain, branched or cyclic arrangement of carbon atomsconnected by single carbon to carbon bonds and/or by ether linkages, andsubstituted accordingly with hydrogen atoms. As used herein,“substituted hydrocarbyl” means a group that is substituted and containsone or more substituent groups that do not cause the substrate,catalyst, or product to be unstable or unsuitable for the use orreaction intended. Substituent groups which are generally useful includeether, ester, halo, amino (including primary, secondary and tertiaryamine groups), hydroxy, vinylidene or substituted vinylidene, silyl orsubstituted silyl, nitro, nitroso, sulfinyl, sulfonyl, sulfonic acidalkali metal salt, boranyl or substituted boranyl, and thioether.

In one embodiment, the amine protectant is a polyamine polymercomprising an effective number of free amine groups. In anotherembodiment, polyamine polymers include copolymers comprising aneffective number of free amine groups Polyamine polymers may range insize from oligomers (typically less than 1,000 Daltons) to highermolecule weight polymers ranging from 1,000 Daltons to 101000,000Daltons, preferably from 11000 Daltons to 2,000,000 Daltons, morepreferably 1,000 Daltons to 1,000,000 Daltons, and more preferably from10,000 Daltons to 1,000,000 Daltons.

In one embodiment, the polyamine polymer is selected from the groupconsisting of 2-amino-2-deoxy-(1

4)-β-D-glucopyranan (i.e. chitosan), polyalkyleneamine polymers havingalkylene moieties of 2 to 12 carbon atoms, polyethylenimine,polyallylamine, polyvinyl alcohol/polyvinylamine copolymers,D-polylysine, L-polylysine, mixtures of D/L polylysine, polyethyleniminecross-linked with glutaraldehyde, and mixtures thereof. In a preferredembodiment, the polyamine polymer is polyethyleneimine polymer (linearor branched) and copolymers comprising polyethylenimine. In anotherpreferred embodiment, the polyamine polymer is polyethyleniminecross-linked with glutaraldehyde. In a further preferred embodiment, theenzyme catalyst is immobilized in or on a particle or bead comprisingpolyethylenimine cross-linked with glutaraldehyde.

The amine protectant may be soluble or insoluble in the aqueous reactionmixture. Soluble amine protectants can be separated from the subsequentproduct mixture using any number of separation methodologies well knownin the art (for example, ion exchange or chromatographic techniques).Depending upon the chemical nature of the soluble amine protectant, oneof skill in the art may separate the amine protectant from the glycolicacid (or ammonium glycolate) product by adjusting the post reactionprocessing conditions to selectively precipitate or adsorb one or morecomponents from the product mixture. For certain applications, it maynot be necessary to remove the soluble amine protectant from the productmixture, especially for applications where high purity glycolic acidand/or ammonium glycolate is not required.

In a preferred embodiment, the amine protectant is substantiallyinsoluble in the aqueous reaction mixture. The use of an insoluble amineprotectant facilitates simple separation/isolation (i.e. filtration,centrifugation, etc.) from the resulting aqueous product mixture. Inanother preferred embodiment the amine protectant is a polyamine polymerthat is insoluble in the aqueous reaction mixture.

In another embodiment, the amine protectant is an insolubleamine-functionalized material and may include amine-functionalizedsupport materials and amine-functionalized polymers. Methods tochemically modify insoluble materials to include at least one primaryamino group are well-known in the art (for example, see Bickerstaff, G.F., Immobilization of Enzymes and Cells, Methods in Biotechnology,Volume 1. 1997. Humana Press, Totowa, N.J., and Wong, S. S., Chemistryof Protein Conjugation and Crosslinking, 1991. CRC Press, Boca Raton,Fla.).

The amine-functionalized material comprising an effective number of freeamine groups (or functionalized to contain an effective number freeamine groups) useful in the present process may include, but is notlimited to polysaccharides that have been chemically functionalized tocontain one or more amino groups (for example, amine-functionalizedglycans, agarose, carrageenan, alginate, dextran, and cellulose),methacrylates, polyurethanes, polyesters, nylons, alumina, silica,polystyrene, polyvinyl alcohols, magnetite, and controlled pore glass.Polyalkyleneamine polymers (typically having alkylene moieties of 2 to12 carbon atoms) and polyalkyleneamine-functionalized support materialsmay be used, as may soluble or insoluble polyethyleneamine polymers,polyethyleneamine-functionalized supports materials, polyethylenimine,polyethylenimine polymers (linear or branched), polyethyleniminecross-linked with glutaraldehyde, polyethylenimine ion exchange resins,weakly basic anion exchange resins (for example, AMBERLITE® weakly basicanion exchange resins IRA-95, IRA-96, IRA-67, and IRA-92 (available fromRohm and Haas, Philadelphia, Pa.), and DIAION® WA20 or WA21J (availablefrom Mitsubishi Chemical, Tokyo, Japan), polyethylenimine-cellulose,polyethyleneimine-silica, polyallylamine, polyvinylalcohol/polyvinylamine copolymers, polylysine (D, L, or D/L mixtures),w-aminohexylagarose, w-aminododecylagarose, w-aminoethylagarose,aminopropylsilated glass beads, and combinations thereof. In a preferredembodiment, the amine protectant does not include a cyano group capableof being hydrolyzed by the nitrilase catalyst.

In one embodiment, amino alkyl-, amino(hydroxyalkyl)-,aminoalkyl-ether-, and amino(hydroxyalkyl)-ether derivatives ofcellulose, chitin and other naturally-occurring carbohydrates arepreferably selected from the group consisting of:

H₂N—(CH₂)_(n)-[carbohydrate] where n=1-10, including alkyl isomers,H₂N—(CH₂)_(m)—CHOH—(CH₂)_(n)-[carbohydrate], where m=0-10 and n=0-10,H₂N—(CH₂)_(n)—O-[carbohydrate] where n=1-10, andH₂N—(CH₂)_(m)—CHOH—(CH₂)_(n)—O-[carbohydrate] where m=0-10 and n=0-10.

In another embodiment, aminophenyl or aminobenzyl derivatives ofcellulose, chitin or other naturally occurring carbohydrates arepreferably selected from the group consisting of:

H₂N—C₆H₄—(CH₂)_(n)-[carbohydrate],H₂N—CH₂—C₆H₄— (CH₂)_(n)-[carbohydrate]H₂N—C₆H₄—(CH₂), —O—[carbohydrate] where n=0-10, andH₂N—C₆H₄—(CH₂)_(m)—CHOH—(CH₂)_(n)—[carbohydrate],wherein m=0-10 and n=0-10, including p-, o- and m-benzene ringamino-isomers, aminomethyl-isomers and alkyl group isomers thereof.

Amine-functionalized materials may additionally be prepared as mixedpolysaccharide-polymeric derivatives wherein primary amine/aminoalkyl(one to ten carbons per alkyl group), aminohydroxyalkyl (one to tencarbons per alkyl group and one to ten hydroxyl groups per alkyl group),aminobenzene and/or aminoalkylbenzene (one to ten carbons per alkylgroup) functional groups are covalently attached to matrices such asepichlorohydrin copolymers of cellulose or chitin and whereinhydrocarbon spacer groups may include alkene as well as alkyl groups.

Non-polysaccharide polymeric derivatives wherein primary amine,aminoalkyl (one to ten carbons per alkyl group), aminohydroxyalkyl (oneto ten carbons per alkyl group and one to ten hydroxyl groups per alkylgroup), aminobenzene and/or aminoalkylbenzene (one to ten carbons peralkyl group) functional groups are covalently attached to any one of awide variety of synthetic polymers including polystyrene,styrene-divinylbenzene copolymer, polyvinyl alcohol and cross-linkedderivatives thereof, and wherein hydrocarbon spacer groups may includealkene as well as alkyl groups.

In another embodiment, the enzyme catalyst is immobilized in a matrixcomprising at least one amine protectant. In another embodiment theenzyme catalyst is immobilized in an insoluble matrix comprising aneffective number of free amine groups. In a preferred embodiment, theinsoluble matrix provides and effective number of primary amine groups(i.e. carrageenan beads crosslinked with glutaraldehyde andpolyethylenimine).

In those applications where an insoluble amine protectant additive neednot be removed from the final product, or where said additive is readilyremoved from the product mixture (for instance by ion exchange ordistillation), then soluble amine protectants may be used. Examples ofsoluble amine protectants may include, but are not limited to solubleforms of the above mentioned insoluble polymers (e.g., soluble lowmolecular monomers), C₁-C₁₂ aliphatic, cycloaliphatic, or aromaticcompounds comprising one or more primary amino groups,1-amino-2-ethanol, 1-amino-2-propanol, Tris buffer, ethylenediamine andsimilar soluble amine protectants comprising at least one primary aminogroup.

In a preferred embodiment, the amine protectant is polyethylenimine. Ina further preferred embodiment, the nitrilase catalyst is immobilized inan insoluble matrix crosslinked with glutaraldehyde andpolyethylenimine.

One of skill in the art can adjust the amount of amine protectant in thereaction mixture to achieve the desired effect (i.e. an improved innitrilase activity; also referred to herein as an “effective amount”).In one embodiment, the concentration of amine protectant is at least0.01 mg/mL, preferably at least 0.1 mg/L, more preferably at least 1mg/mL, and most preferably at least 5 mg/mL.

Hydrolysis of Glycolonitrile to Glycolic Acid Using a Nitrilase Catalyst

The enzymatic conversion of glycolonitrile to glycolic acid (in the formof the acid and/or the corresponding ammonium salt) was performed bycontacting an enzyme catalyst (comprising a polypeptide having nitrilaseactivity) with a suitable aqueous reaction mixture comprisingglycolonitrile using a suitable set of enzymatic reaction conditions (pHrange, temperatures, concentrations, etc.) described below. In oneembodiment, whole recombinant microbial cells can be used as an enzymecatalyst without any pretreatment. In another embodiment, the microbialcell catalyst can be added directly to a reaction mixture, or maintainedseparately from the bulk reaction mixture using hollow-fiber membranecartridges or ultrafiltration membranes. In a further embodiment themicrobial cells can be immobilized in a polymer matrix (e.g.,carrageenan or polyacrylamide gel (PAG) particles) or on an insolublesolid support (e.g., celite) to facilitate recovery and reuse of theenzyme catalyst (U.S. Pat. No. 6,870,038; herein incorporated byreference). In yet a further embodiment, purified or partially-purifiedenzyme(s) can also be isolated from the whole cells and used directly asa catalyst, or the enzyme(s) can be immobilized in a polymer matrix oron an insoluble support. Methods for the immobilization of cells or forthe isolated enzymes have been widely reported and are well known tothose skilled in the art (Methods in Biotechnology, Vol. 1Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor;Humana Press, Totowa, N.J., USA; 1997). The immobilization of the A.facilis 72W nitrilase catalyst has previously been reported (U.S. Pat.No. 6,870,038).

The concentration of enzyme catalyst in the aqueous reaction mixturedepends on the specific activity of the enzyme catalyst and is chosen toobtain the desired rate of reaction. The wet cell weight of themicrobial cells used as catalyst in hydrolysis reactions typicallyranges from 0.001 grams to 0.250 grams of wet cells per mL of totalreaction volume, preferably from 0.002 grams to 0.050 grams of wet cellsper mL.

The temperature of the glycolonitrile hydrolysis reaction is chosen tocontrol both the reaction rate and the stability of the enzyme catalystactivity. The temperature of the reaction may range from just above thefreezing point of the reaction mixture (approximately 0° C.) to about65° C., with a preferred range of reaction temperature of from about 5°C. to about 35° C. The microbial cell catalyst suspension may beprepared by suspending the cells in distilled water, or in a aqueoussolution of a buffer which will maintain the initial pH of the reactionbetween about 5.0 and about 10.0, preferably between about 5.5 and about8.0, more preferably between about 5.5 and about 7.7, and mostpreferably about 6.0 to about 7.7. As the reaction proceeds, the pH ofthe reaction mixture may change due to the formation of an ammonium saltof the carboxylic acid from the corresponding nitrile functionality. Thereaction can be run to complete conversion of glycolonitrile with no pHcontrol, or a suitable acid or base can be added over the course of thereaction to maintain the desired pH.

Glycolonitrile was found to be completely miscible with water in allproportions at 25° C. In cases where reaction conditions are chosen suchthat the solubility of the substrate (i.e., an α-hydroxynitrile) is alsodependent on the temperature of the solution and/or the saltconcentration (buffer or product glycolic acid ammonium salt, also knownas ammonium glycolate) in the aqueous phase, the reaction mixture mayinitially be composed of two phases: an aqueous phase containing theenzyme catalyst and dissolved α-hydroxynitrile, and an organic phase(the undissolved α-hydroxynitrile). As the reaction progresses, theα-hydroxynitrile dissolves into the aqueous phase, and eventually asingle phase product mixture is obtained. The reaction may also be runby adding the α-hydroxynitrile to the reaction mixture at a rateapproximately equal to the enzymatic hydrolysis reaction rate, therebymaintaining a single-phase aqueous reaction mixture, and avoiding thepotential problem of substrate inhibition of the enzyme at high startingmaterial concentrations.

Glycolic acid may exist in the product mixture as a mixture of theprotonated carboxylic acid and/or its corresponding ammonium salt(dependent on the pH of the product mixture; pKa of glycolic acid isabout 3.83), and may additionally be present as a salt of the carboxylicacid with any buffer that may additionally be present in the productmixture. Typically, the glycolic acid produced is primarily in the formof the ammonium salt (pH of the glycolonitrile hydrolysis reaction istypically between about 5.5 and about 7.7). The glycolic acid productmay be isolated from the reaction mixture as the protonated carboxylicacid, or as a salt of the carboxylic acid, as desired.

The final concentration of glycolic acid in the product mixture atcomplete conversion of glycolonitrile may range from 0.001 M to thesolubility limit of the glycolic acid product. In one embodiment, theconcentration of glycolic acid will range from about 0.10 M to about 5.0M. In another embodiment, the concentration of glycolic acid will rangefrom about 0.2 M to about 3.0 M.

Glycolic acid may be recovered in the form of the acid or correspondingsalt using a variety of techniques including, but not limited to ionexchange, electrodialysis, reactive solvent extraction, polymerization,thermal decomposition, alcoholysis, and combinations thereof (seeco-pending U.S. Patent Application Publication No. 2006-0247467).

Microbial Expression

The nitrilase catalyst may be produced in heterologous host cells,preferably in microbial hosts. Particularly useful in the presentinvention will be cells that are readily adaptable to large-scalefermentation methods. Such organisms are well known in the art ofindustrial bioprocessing, examples of which may be found in RecombinantMicrobes for Industrial and Agricultural Applications, Murooka et al.,eds., Marcel Dekker, Inc., New York, N.Y. (1994), and includefermentative bacteria as well as yeast and filamentous fungi. Host cellsmay include, but are not limited to Comamonas sp., Corynebacterdum sp.,Brevibacterium sp., Rhodococcus sp., Azotobacter sp., Citrobacter sp.,Enterobacter sp., Clostridium sp., Klebsiella sp., Salmonella sp.,Lactobacillus sp., Aspergillus sp., Saccharomyces sp., Yarrowia sp.,Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp., Candida sp.,Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp., Torulopsissp., Methylobacteda sp., Bacillus sp., Escherichia sp., Pseudomonas sp.,Rhizobium sp., and Streptomyces sp. Particularly preferred is E. coli.Examples of suitable E. coli host cells in which a mutant nitrilase genecan be expressed include, but are not limited to, host cells specifiedherein and MG1655 (ATCC 47076), FM5 (ATCC 53911), W3110 (ATCC 27325),MC4100 (ATCC 35695), W1485 (ATCC 12435), and their derivatives. Inanother aspect the preferred E. coli host strains are MG1655 (ATCC47076) or FM5 (ATCC 53911),

Heterologous expression of the A. facilis 72W nitrilase has previouslybeen reported (Chauhan et al., supra; U.S. Pat. No. 6,870,038; U.S. Pat.No. 7,148,051; and U.S. Pat. No. 7,198,927). Chauhan et al. report an E.coli strain (E. coli SS1001 (ATCC PTA-1177)) that expressed active A.facilis 72W nitrilase (SEQ ID NO: 57). The coding sequence of therecombinantly expressed (E. coli SS1001) nitrilase contained two minorsequence changes in comparison to the wild-type 72W nitrilase sequence(SEQ ID NOs: 3 and 4). The start codon was changed from GTG to ATG tofacilitate recombinant expression and an artifact was introduced duringcloning that resulted in a single amino acid change near the C-terminal(Pro367 [CCA]

Ser [TCA]).

Recombinant expression in an industrially-suitable host has severaladvantages. First, the genetic toolbox for many of the commonly usedproduction hosts is usually well developed in comparison to the genetictools available for many of the microorganisms from which the gene ofinterest was obtained. Recombinant expression in these hosts is normallymore cost effective than expression in the native host. For example, ithas been shown that A. facilis 72W cells grow on glycerol, a relativelyexpensive carbon substrate, when grown by fermentation, and have notbeen successfully grown using inexpensive glucose. In contrast, E. colitransformants can be grown on glucose to the same cell density as A.facilis 72W cells in about half the time, significantly reducingbiocatalyst production costs (U.S. Pat. No. 6,870,038).

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well-known to those skilled in the art. These could be usedto construct chimeric genes for production of the gene products. Thesechimeric genes could then be introduced into appropriate microorganismsvia transformation to provide high level expression of the desirednitrilase.

Chimeric genes will be effective in altering the properties of a hostcell. For example, introducing at least one copy of chimeric genesencoding the present nitrilases under the control of the appropriatepromoters into a host cell gives the host cell an improved ability toconvert glycolonitrile to glycolic acid. The chimeric genes willcomprise suitable regulatory sequences useful for driving geneexpression of the present mutant nitrilase sequences. Suitableregulatory sequences may include, but are not limited to promoters,translation leader sequences, and ribosomal binding sites. It ispreferred if these sequences are derived from the host organism;however, the skilled person will recognize that heterologous regulatorysequences may also be used.

Chimeric genes can be introduced into an appropriate host by cloning itinto a suitable expression vector. Vectors or cassettes useful for thetransformation of suitable host cells are well known in the art.Typically, the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the codingsequence that harbors transcriptional initiation controls and a region3′ of the DNA fragment which controls transcriptional termination. It ismost preferred when both control regions are derived from geneshomologous to the host cell, although such control regions need not bederived from the genes native to the specific species chosen as aproduction host.

In one embodiment, the regulatory sequences will include a promoter.Promoters may be constitutive or inducible. Inducible promoters aregenerally responsive to a specific stimulus (e.g., IPTG or lactoseinducing the lac promoter). Inducible promoters may be responsive to avariety of stimuli, including, chemicals, growth cycle, changes intemperature, changes in pH and changes in osmolarity, to name only afew.

Initiation control regions or promoters that are useful to driveexpression of the nitrilase in the desired host cell are numerous andfamiliar to those skilled in the art, including but not limited to CYC1,HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI (useful for expression in Saccharomyces); AOX1 (useful forexpression in Pichia); and lac, trp, IP_(R), IP_(R), T7, tac, P_(BAD),npr, and trc (particularly useful for expression in Escherichia colt).Additional examples of promoters particularly suitable for drivingexpression in E. coli include, but are not limited to the tryptophanoperon promoter Ptrp of E. coli, a lactose operon promoter Plac of E.coli, a Ptac promoter of E. coli, a phage lambda right promoter P_(R), aphage lambda left promoter P_(L), a T7 promoter, and a promoter of theGAP gene from Pichia pastors, or is at least one promoter isolated fromthe group of microorganisms selected from the group consisting ofComamonas, Corynebacterdum, Brevibacterium, Rhodococcus, Azotobacter,Citrobacter, Enterobacter, Clostridium, Klebsiella, Salmonella,Lactobacilus, Aspergillus, Saccharomyces, Pichia, Zygosaccharomyces,Kluyveromyces, Candida, Hansenula, Dunaliella, Debaryomyces, Mucor,Torulopsis, Methylobacteria, Bacillus, Escherichia, Pseudomonas,Rhizobium, and Streptomyces.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included.

Additionally, the inserted genetic material may include a ribosomebinding site (RBS). The ribosome binding site may be from a phage lambdaCII gene or is selected from the group consisting of ribosome bindingsites from a gene of Comamonas, Corynebacterium, Brevibacterium,Rhodococcus, Azotobacter, Citrobacter, Enterobacter, Clostridium,Klebsiella, Salmonella, Lactobacillus, Aspergillus, Saccharomyces,Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,Dunaliella, Debaryomyces, Mucor, Torulopsis, Methylobacteria, Bacillus,Escherichia, Pseudomonas, Rhizobium, and Streptomyces.

Optionally, the gene products may preferably be a secreted product ofthe transformed host. Secretion of desired proteins into the growthmedia simplifies purification procedures and reduces costs. Secretionsignal sequences are often useful in facilitating the active transportof expressible proteins across cell membranes. A transformed hostcapable of secretion may be created by incorporating in the host a DNAsequence that codes for a secretion signal. Methods for choosingappropriate signal sequences are well known in the art (see for exampleEP 546049; WO 93/24631). The secretion signal DNA may be located betweenthe expression-controlling DNA and the instant coding sequence or codingsequence fragment, and in reading frame with the latter.

Industrial Production of the Microbial Catalyst

Where commercial production of the nitrilase catalyst is desired, avariety of culture methodologies may be used. Fermentation runs may beconducted in batch, fed-batch, or continuous mode, methods well-known inthe art (Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass., (1989); Deshpande, Mukund V., Appl. Biochem.Biotechnol. 36(3): 227-234 (1992)).

A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to artificial alterations during the culturing process. Thus, atthe beginning of the culturing process the media is inoculated with thedesired organism or organisms and growth or metabolic activity ispermitted to occur adding nothing to the system. Typically, however, a‘batch’ culture is batch with respect to the addition of carbon sourceand attempts are often made at controlling factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch culture processes are also suitable in the present inventionand comprise a typical batch system with the exception that thesubstrate is added in increments as the culture progresses. Fed-Batchsystems are useful when catabolite repression is apt to inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the media, Measurement of the actual substrateconcentration in Fed-Batch systems is difficult and is thereforeestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen, and the partial pressure of waste gases such as CO₂.Batch and Fed-Batch culturing methods are common and well known in theart and examples may be found in Brock (supra) and Deshpande (supra).

Commercial production of the nitrilase catalysts may also beaccomplished with a continuous culture. Continuous cultures are an opensystem where a defined culture media is added continuously to abioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous cultures generally maintainthe cells at a constant high-liquid-phase density where cells areprimarily in log phase growth. Alternatively, continuous culture may bepracticed with immobilized cells where carbon and nutrients arecontinuously added and valuable products, by-products or waste productsare continuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

Continuous or semi-continuous culture allows for the modulation of onefactor or any number of factors that affect cell growth or end cellconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen level at a fixed rate and allowall other parameters to moderate. In other systems a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady-state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes, as well as techniques formaximizing the rate of cell formation, are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock(supra).

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include, but are not limited tomonosaccharides such as glucose and fructose, disaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof, and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Hence, it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing substrates and will only be limited by the choice oforganism.

Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given either as a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

General Methods

The following examples are provided to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (1994) (Phillipp Gerhardt, R. G. E.Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R.Krieg and G. Briggs Phillips, eds.), American Society for Microbiology,Washington, D.C.) or by Thomas D. Brock, in Biotechnology: A Textbook ofIndustrial Microbiology, (1989) Second Edition, (Sinauer Associates,Inc., Sunderland, Mass.). Methods to immobilize enzymatic catalysts canbe found in Bickerstaff, G. F., supra).

Procedures required for genomic DNA preparation, PCR amplification, DNAmodifications by endo- and exo-nucleases for generating desired ends forcloning of DNA, ligations, and bacterial transformation are well knownin the art. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by Maniatis,supra; and by T. J. Silhavy, M. L. Bennan, and L. W, Enquist,Experiments with Gene Fusions, (1984) Cold Spring Harbor LaboratoryPress, Cold Spring, N.Y.; and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, (1994-1998) John Wiley & Sons, Inc., NewYork.

All reagents and materials were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma/Aldrich Chemical Company (St. Louis, Mo.)unless otherwise specified.

The abbreviations in the specification correspond to units of measure,techniques, properties, or compounds as follows: “sec” means second(s),“min” means minute(s), “h” or “hr” means hour(s), “d” means density ing/mL, “μL” means microliters, “mL” means milliliters, “L” means liters,“mM” means millimolar, “M” means molar, “mw” means average moleculeweight, “mmol” means millimole(s), “wt” means weight, “wt %” meansweight percent, “g” means grams, “μg” means micrograms, HPLC” means highperformance liquid chromatography, “O.D.” means optical density at thedesignated wavelength, “ddw” means dry cell weight, ‘U’ means units ofnitrilase activity, “EDTA” means ethylenediaminetetraacetic acid, “DTT”means dithiothreitol, “DI” means distilled and deionized”, and “NCIMB”means National Collections of Industrial Food and Marine Bacteria,Bucksburn, Aberdeen, Scotland, UK. One U of nitrilase activitycorresponds to the hydrolysis of 1 μmol glycolonitrile/min.

Analytical Methodology HPLC Analysis

Unless otherwise noted, the following HPLC method was used. The reactionproduct mixtures were analyzed by the following HPLC method. Aliquots(0.01 mL) of the reaction mixture were added to 1.50 mL of water, andanalyzed by HPLC (HPX 87H column, 30 cm×7.8 mm; 0.01 NH₂SO₄ mobilephase; 1.0 mL/min flow at 50 (C; 10 μL injection volume; RI detector, 20min analysis time). The method was calibrated for glycolonitrile at aseries of concentrations using commercially available glycolonitrilepurchased from Aldrich.

Example 1 Dependence of Nitrilase Specific Activity of Unimmobilized E.coli MG1655/pSW138-168V on Added Polyethylenimine

To a 20-mL glass vial with magnetic stir bar was added 1.0 mL of a 25.0mg (dry cell wt.)/mL suspension of E. coli MG1655/pSW138-168V (SEQ IDNO: 51) in 0.1 M KH₂PO₄ buffer (pH 7.5), and either 1.0 ml of 0.3 MKH₂PO₄ buffer (pH 7.5) or 1.0 mL of a 10.2 mg/mL solution ofpolyethylenimine (BASF LUPASOL® PS, 750,000 mw; BASF Aktiengesellschaft,Ludwigshafen, Germany) in 0.3 M KH₂PO₄ buffer (pH 7.5) at 25° C. Withstirring, 2.0 mL of an aqueous solution containing glycolonitrile (1.0M) and formaldehyde (8 mM, 1 mM, or 0.04 mM) in distilled, deionizedwater at 25° C. was added, and the reaction mixture maintained at 25° C.with a temperature bath. At 5, 10, 15, and 30 minutes, a 100 μl aliquotof the reaction mixture was removed and mixed with 100 μl of water, 10μl of 6.0 N HCl and 200 μl of 0.25 M n-propanol in water (HPLC externalstandard), the mixture centrifuged, and the resulting supernatantanalyzed by HPLC to determine the initial reaction rate and catalystspecific activity (U/g dcw) (Table 2).

TABLE 2 Dependence of biocatalyst specific activity on concentration offormaldehyde with and without added polyethylenimine (PEI).glycolonitrile formaldehyde PEI specific activity (mM) (mM) (mg/mL) (U/gdcw) 500 4.0 0 471 500 4.0 5.1 1484 500 0.50 0 1344 500 0.50 5.1 1726500 0.02 0 1742 500 0.02 5.1 1629

Example 2 Preparation of Copoly(Vinylalcohol/Vinylamine)

A solution of 0.2 g sodium dodecylbenzenesulfonate (Sigma-AldrichCatalog #289957) and 0.2 g sodium dihydrogen phosphate in 80 mLdeionized water was placed in a 250-mL, 4-neck RB flask with condenserand nitrogen inlet, thermometer, dropping funnel and magnetic stirrer.The flask was swept with nitrogen and was stirred in a 72° C. water bathuntil the solution temperature was 65° C.; then 0.1 g VAZO®-64(2,2′-azobisisobutyronitrile; mw: 164.2; Sigma-Aldrich catalog #441090)initiator was added.

A solution of 40 g vinyl acetate (Sigma-Aldrich catalog #V1503, filteredthrough basic alumina to remove inhibitor), 4 g N-vinylformamide(Sigman-Aldrich catalog #447331, used without further purification) and0.3 g VAZO® 64 was placed in the dropping funnel and 5 mL of thismonomer solution was added to the flask. The mixture was stirred 20 minand another 5 mL of monomer was added. Five-mL aliquots of monomer wereadded every 20 min until 20 mL had been added (at 1 hr); then themixture was stirred at 70° C. for 1 hr. After this the remainder of themonomer was added at a rate of 5 mL every 20 min. Monomer addition wascomplete at 4 h, and then the mixture was stirred at 70° C. for 3 h andallowed to cool to room temperature (approximately 22° C.). Theresulting polymer beads were washed several times with hot water andthen allowed to stand in water for 72 hr. The beads were then washedwith diethyl ether and allowed to stand in ether for 15 min to extractvinyl monomers, followed by filtration and drying under vacuum with anitrogen blanket, then dried overnight at 70° C. in a vacuum oven at 350mm Hg with a nitrogen sweep to yield 39.0 g ofpoly(vinylacetate-vinlyformamide)copolymer.

Poly(vinylacetate-vinlyformamide)copolymer (25.1 g) was stirred atreflux with 200 mL methanol containing 6 mL concentrated HCl and 5 mLwater for 5 h, yielding a clear solution which was allowed to stand atRT overnight. The solution became a rubbery gel; this was blended withseveral changes of acetone to precipitate the hydrolyzed polymer HClsalt, which was dried under nitrogen in the vacuum oven at 75° C.(yield: 15 g). Analysis by ¹H NMR (DMSO-d6) indicated about 20% of theacetates remained unhydrolyzed, so the product was stirred at refluxwith a mixture of 150 mL methanol and 5 mL conc. HCl overnight tocomplete hydrolysis. The methanol was filtered off, and the rubbery,methanol-insoluble product was stirred with 100 mL Dl water at 90° C.;this solution was filtered first through a screen to remove a smallamount of gel and then through a Millipore cellulose prefilter(Millipore Corp., Bedford, Mass.) under pressure. The filtered solutionwas basified to pH 9.0 with 10% NaOH and then dialyzed overnight againstDl water in a MEMBRA-CEL® 3.5K molecular weight cut-off dialysismembrane tube (Viskase Co., Willowbrooke, Ill.). Lyophilization yielded8.7 g of copoly(vinylalcohol/vinylamine).

Example 3 Dependence of Nitrilase Specific Activity of Unimmobilized E.coli MG1655/pSW138-168V on Added Amine Protectants

To a 20-mL glass vial with magnetic stir bar was added 20 mg to 400 mgof amine protectant (see Table 2), followed by 1.825 mL of deionizedwater and 1.0 mL of 0.3 M KH₂PO₄ buffer (pH 7.5). The pH of theresulting mixture was checked and re-adjusted to pH 7.5 with 6 N HCl.When the pH of the resulting mixture was stable at pH 7.5, 10 mL of aca. 25.0 mg (dry cell wt.)/mL suspension of E. coli MG 1655/pSW138-168V(SEQ ID NO: 51) in 0.1 M KH₂PO₄ buffer (pH 7.5) was added, then thereaction was initiated by the addition of 0.175 mL (0.187 g) of aqueousglycolonitrile (61 wt % GLN in water, 2.00 mmol of glycolonitrile insolution also containing either 0.80 mol % or 0.10 mol % formaldehyderelative to glycolonitrile), and the reaction mixture maintained at 25°C. with a temperature bath. At 5, 10, 15, and 30 minutes, a 100 Mlaliquot of the reaction mixture was removed and mixed with 100 μl ofwater, 10 Fl of 6.0 N HCl and 200 μl of 0.25 M n-propanol in water (HPLCexternal standard), the mixture centrifuged, and the resultingsupernatant analyzed by HPLC to determine the initial reaction rate andcatalyst specific activity (U/g dcw) (Table 3). Each set of experimentslisted in Table 3 were performed with a freshly-prepared cell suspension(cell suspensions A-G), where the differences in the specific activityof the cells in the control reaction for each cell suspension were dueto differences in the dry cell weight concentrations of the individualcell suspensions.

TABLE 3 Dependence of biocatalyst specific activity on concentration offormaldehyde with and without added amine protectant. amine specificcell glycolonitrile formaldehyde protectant activity suspension (mM)(mM) amine protectant (mg/mL) (U/g dcw) A 500 4.0 PEI/silica 100 491(Aldrich 24675-1) A 500 4.0 PEI/cellulose 100 1212 (Sigma P6883) A 5004.0 none (control) 0 385 B 500 4.0 3-aminopropyl 100 799 silica gel(Sigma A1409) B 500 4.0 none (control) 0 481 C 500 4.0 Long-chain alkyl100 1136 amine controlled pore glass (Sigma L8638) C 500 4.0 none(control) 0 527 D 500 0.5 Long-chain alkyl 100 1891 amine controlledpore glass (Sigma L8638) D 500 0.5 none (control) 0 1325 E 500 4.0polyallylamine 5 598 (Aldrich 47914-4) E 500 4.0 polyallylamine 10 648(Aldrich 47914-4) E 500 4.0 PVOH/poly- 70 1541 vinylamine (Example 2) E500 4.0 Poly-D-lysine 11 502 (Sigma P0296) E 500 4.0 none (control) 0464 F 500 0.5 PVOH/poly- 70 1786 vinylamine (Example 2) F 500 0.5Poly-D-lysine 11 1388 (Sigma P0296) F 500 0.5 none (control) 0 1220 G500 4.0 DIAION ® WA20 100 1128 (Mitsubishi Chemical) G 500 4.0 DIAION ®WA21J 100 1073 (Mitsubishi Chemical) G 500 4.0 none (control) 0 508 G500 0.5 DIAION ® WA20 100 1335 (Mitsubishi Chemical) G 500 0.5 DIAION ®WA21J 100 1339 (Mitsubishi Chemical) G 500 0.5 none (control) 0 1182

Example 4 Preparation of GA/PEI-Crosslinked Carrageenan/E. coliMG1655/pNM18-168V Beads

With rapid stirring, 12 g of carrageenan (FMC GP911; FMC Corp.,Philadelphia, Pa.) was slowly added to 228 g deionized distilled waterat 50° C., the resulting mixture heated to 80° C. until the carrageenanwas completely dissolved, and the resulting solution cooled withstirring to 52° C. In a separate beaker equipped with stir bar, 83.2 gof frozen E. coli MG1655/pNM18-168V (SEQ ID NO: 51) cells (25.2% dcw)was added to 84.8 g of 0.35 M Na₂HPO₄ (pH 7.3) at ca. 25° C. and mixeduntil the cells were suspended, then a deoxyribonuclease I solution (10μL of 12,500 U/mL DNase (Sigma-Aldrich)/100 mL of cell suspension) wasadded. The cell suspension was filtered consecutively through a 230micron and 140 micron NUPRO® TF strainer (Swagelok Company, Solon, Ohio)element filter, and heated with stirring to 50° C. With stirring, 160.0g of E. coli E. coli MG1655/pNM18-168V cell suspension at 50° C. wasadded to the carrageenan solution at 52° C., and the resultingcell/carrageenan suspension was pumped through an electrically-heated 20gauge needle at 47° C. and dripped into 0.25 M KHCO₃ (pH=7.3) withstirring at ca. 37-38° C.); the flow rate through the needle was set at5-8 mL/min. The resulting beads were allowed to harden in this samebuffer for 1 h at room temperature with stirring, and were stored in0.25 M potassium bicarbonate (pH 7.3).

Chemical cross-linking of a portion of the immobilized cell/carrageenanbeads was performed by addition of 1.0 g of 25% glutaraldehyde (GA) inwater (Sigma M 752-07) to 20 g beads suspended in 48 mL of 0.25 Mpotassium bicarbonate (pH 7.3), and stirring for 1 h at roomtemperature. To the suspension of beads was then added 4.0 g of 12.5 wt% polyethylenimine (PEI, BASF LUPASOL® PS) in water, and the beadsuspension stirred for an additional 18 h at room temperature. TheGA/PEI-crosslinked beads were recovered from the suspension, stirredtwice for 15 min in 48 mL of 0.25 M potassium bicarbonate (pH 7.3), thenstored in 1.0 M ammonium bicarbonate (pH 7.3) at 5° C. Prior to use, thebeads were washed twice for 15 min with 180 mL of 0.1 M ammoniumglycolate (pH 7.0) at room temperature.

Example 5 Dependence of Biocatalyst Specific Activity onGlutaraldehyde/polyethylenimine Cross-linking of Carrageenan-immobilizedE. coli MG1655/pSW138-168V Transformants Expressing A. facilis 72WNitrilase

In a typical procedure, a 50-mL jacketed reaction vessel equipped withoverhead stirring and temperature control was charged with 4.0 g ofGA/PEI cross-linked E. coli MG1655/pSW138-168V/carrageenan beads(prepared using the process as described in Example 4) containing 5%(dcw) transformant expressing the A. facilis 72W nitrilase mutant F168V(SEQ ID NO: 51). To the vessel was then added 10.85 mL of distilledwater and 3.0 mL of aqueous ammonium glycolate (4.0 M, pH 7.0). Themixture was stirred at 25° C. while 1.75 mL of 60.8 wt % glycolonitrile(GLN) in water (1.876 g, 20.0 mmol GLN, 0.160 mmol formaldehyde;stabilized with 0.7 wt % glycolic acid)) and 0.40 mL of aqueous ammoniumhydroxide (1.875 wt % NH₃) was added simultaneously (final pH 7.5). Four0.050-mL reaction samples were removed at pre-determined times after theGLN addition and analyzed by HPLC to determine the initial reaction rateand the catalyst specific activity (μmol glycolic acid/min/g dcwbiocatalyst).

TABLE 4 Dependence of immobilized biocatalyst specific activity onglutaraldehyde/polyethylenimine cross-linking. specific specificglycolonitrile formaldehyde GA/PEI activity activity (mM) (mM)cross-linked (U/g beads) (U/g dcw) 1000 8.0 no 36.2 723 1000 8.0 yes68.2 1364 1000 1.0 no 58.4 1169 1000 1.0 yes 99.7 1994 1000 0.04 no 68.81375 1000 0.04 yes 90.1 1801

Example 6 Comparison of Biocatalyst Specific Activity for Uncross-Linkedand Glutaraldehyde/Polyethylenimine Cross-Linked Carrageenan-ImmobilizedE. coli MG1655/pSW138-168V Transformants Expressing A. facilis 72WNitrilase in Consecutive Batch Reactions with Catalyst Recycle

A 50-mL jacketed reaction vessel equipped with overhead stirring andtemperature control was charged with 8 g of GA/PEI-crosslinked E. coliMG1655/pSW138-168V/carrageenan beads (prepared using the process asdescribed in Example 4) containing 5% (dcw) transformant expressing theA. facilis 72W nitrilase mutant F168V (SEQ ID NO: 51). To the vessel wasthen added 14.78 mL of distilled water and 6.0 mL of aqueous ammoniumglycolate (4.0 M, pH 7.0), and the reaction vessel flushed withnitrogen. The mixture was stirred at 25° C. while programmable syringepumps were used to simultaneously add 1.07 mL of 60 wt % glycolonitrile(GLN) in water (12.0 mmol GLN, 0.084 mmol formaldehyde; Fluka(redistilled, stabilized with 0.5 wt % glycolic acid, available fromSigma-Aldrich) and 0.150 mL of aqueous ammonium hydroxide (1.875 wt %);one equivalent volume of GLN and ammonium hydroxide solutions was addedsimultaneously every 2 h to maintain the concentration of GLN at ≦400 mMand the pH within a range of 6.5-7.5. Four 0.050-mL reaction sampleswere removed at pre-determined times after the first GLN addition andanalyzed by HPLC to determine the initial reaction rate and the catalystspecific activity (μmol glycolic acid/min/g dcw biocatalyst). Atcompletion of the reaction, there was 100% conversion of GLN to produceglycolic acid (as the ammonium salt) in >99% yield, and theconcentration of ammonium glycolate produced from added GLN wasapproximately 2.5 M (3.1 M total ammonium glycolate when includinginitial ammonium glycolate buffer in a final reaction volume of ca. 38.0mL).

At the end of the first reaction, the aqueous product mixture wasdecanted from the catalyst (under nitrogen), leaving ca. 10.3 g of amixture of immobilized cell catalyst (8.0 g) and remaining productmixture (ca. 2.3 g). To the reaction vessel then added 20.78 mL ofdistilled, deionized water, and a second reaction was performed at 25°C. by the addition of aliquots of aqueous GLN and ammonium hydroxide asdescribed immediately above. The specific activity of recoveredbiocatalyst in consecutive batch reactions with catalyst recycle arelisted in Table 5. To compare the effect ofglutaraldehyde/polyethylenimine cross-linking of carrageenan-immobilizedcells on specific activity, a second series of recycle reactions wasperformed as described above, except that the E. coliMG1655/pSW138-168V/carrageenan beads were not chemically-crosslinkedwith glutaraldehyde and polyethylenimine (Table 5).

TABLE 5 Dependence of biocatalyst specific activity onglutaraldehyde/PEI cross-linking of carrageenan-immobilized microbialcatalyst in consecutive batch reactions with catalyst recyclebiocatalyst specific activity (U/g dcw) with without Reaction GA/PEIGA/PEI Number cross-linking cross-linking 1 1312 215 2 871 385 3 897 2704 816 5 898 296 6 697 7 663 8 792 9 732 10 760 393 11 539 12 495 373 13396 14 560 15 324 443 16 426 17 500 366 18 722 394 19 489 20 21 283 22444 23 325 24 25 501 26 508 285 27 487 224 28 349 29 371 30 345 224

Example 7 Dependence of Nitrilase Specific Activity of UnimmobilizedRhodococcus sp. (ATCC 39484™) on Added Polyethylenimine

A lyophilized culture of Rhodococcus sp. ATCC 39484 (SEQ ID NO: 25) wassuspended in 5 ml of nutrient broth (Difco; catalog #0003-01-6) in a50-mL conical test tube and revived following incubation at 30° C. for 3days at 60 rpm, followed by storage at −80° C. after addition ofglycerol (10% v/v). The revived culture (1 mL) was inoculated into a125-mL unbaffled shake flask containing 50 mL of Miller's Luria-Bertanibroth (Mediatech, Inc.; 46-050-CM; Manassas, Va.) and grown for 16 h at30° C. and 200 rpm to an OD of 9.0 at 600 nm, then glycerol (10% v/v)was added and the inoculum stored at −80° C. The inoculum (2 mL) wastransferred to a 1-L unbaffled shake flask containing 200 mL of a mediumconsisting of 1 g polypeptone (Becton-Dickinson; catalog #11910;Franklin Lakes, N.J.), 0.6 g malt extract (Difco; 0186-02-4), 0.6 gyeast extract (Difco; 0127-17), 2 g glycerol, and 0.2 mL ofisovaleronitrile (T. Nagasawa, M. Kobayashi, H. Yamada, Archives ofMicrobiology, (1988) 150:89-94) at pH 7.0, followed by growth at 30° C.with 200 rpm agitation. At 50 hr and 70 hr the culture was fed with 0.1%(v/v) and 0.2% (v/v) of isovaleronitrile, respectively. The cell pastewas harvested at 96 h by centrifugation at 13,000×g (5° C.) and storedat −80° C.

To a 20-mL glass vial with magnetic stir bar was added 1.0 mL of a 73.0mg (dry cell wt.)/mL suspension of Rhodococcus sp. (ATCC 39484™) in 0.1M KH₂PO₄ buffer (pH 7.5), and either 1.0 ml of 0.3 M KH₂PO₄ buffer (pH7.5) or 1.0 mL of a 10.2 mg/mL solution of polyethylenimine (BASFLUPASOL® PS, 750,000 mw) in 0.3 M KH₂PO₄ buffer (pH 7.5) at 25° C. Withstirring, 2.0 mL of an aqueous solution containing glycolonitrile (1.0M) and formaldehyde (8 mM or 0.04 mM) in distilled, deionized water at25° C. was added, and the reaction mixture maintained at 25° C. At 5,10)₁₋₅, and 30 minutes, a 100 μl aliquot of the reaction mixture wasremoved and mixed with 100 μl of water, 10 μl of 6.0 N HCl and 200 μl of0.25 M n-propanol in water (HPLC external standard), the mixturecentrifuged, and the resulting supernatant analyzed by HPLC to determinethe initial reaction rate and catalyst specific activity (U/g dcw)(Table 6).

TABLE 6 Dependence of Rhodococcus sp. (ATCC 39484 ™) nitrilase specificactivity on concentration of formaldehyde with and without addedpolyethylenimine (PEI). glycolonitrile formaldehyde PEI specificactivity (mM) (mM) (mg/mL) (U/g dcw) 500 4.0 0 598 500 4.0 5.0 780

Example 8 Dependence of Nitrilase Specific Activity of UnimmobilizedRhodococcus rhodochrous (NCIMB 11216) on Added Polyethylenimine

Rhodococcus rhodochrous (NCIMB 11216; SEQ ID NO: 24) was revivedfollowing suspension of the preserved culture in 100 μL of normalsaline, followed by streaking of the normal saline cell suspension ontoplates containing nutrient agar (Difco catalog #0001-01-8) supplementedwith yeast extract (2 g/L; Difco catalog #0127-17) and sodium chloride(5 g/L). The sequence of the nitrilase from R. rhodochrous NCIMB 11216has been reported (U.S. Patent Application Publication No. 2003/0157672and GENBANK® Accession No. CAC88237; SEQ ID NO: 24) The plates wereincubated at 30° C. for 48 h and colonies were inoculated into aqueousE2 basal medium supplemented with adipic acid (0.8 wt %; carbon source)and propionitrile (0.2 wt %; nitrogen source) and incubated for 48 h at30° C. with 200 rpm agitation. The E2 basal medium consisted of: KH₂PO₄,1.4 g/L; NaH₂PO₄, 0.69 g/L; Sodium citrate, 0.1 g/L; CaCl₂.2H₂O, 0.025g/L; KCl, 0.5 g/L; NaCl, 1.0 g/L, MgSO₄.7H₂O, 0.5 g/L; FeSO₄.7H₂O, 0.05g/L; CoCl₂.6H₂O, 0.01 g/L; MnCl₂.4H₂O, 0.001 g/L; ZnCl₂, 0.0005 g/L;NaMoO₄.2H₂O, 0.0025 g/L; NiCl₂.6H₂O, 0.01 g/L; CuSO₄.2H₂O, 0.005 g/L;Biotin, 0.0002 g/L; Folic Acid, 0.0002 g/L; Pyridoxine-HCl, 0.001 g/L;Riboflavine, 0.0005 g/L; Thiamine•HCl, 0.00005 g/L; Nicotinic Acid,0.0005 g/L; Pantothenic Acid, 0.0005 g/L; Vitamin B₁₂, 0.00001 g/L,p-Aminobenzoic Acid, 0.0005 g/L. Following 48 h of growth, 1.59 g of wetcell paste was harvested by centrifugation at 13,000×g, and theresulting cell paste was stored at −80° C.

To a 20-mL glass vial with magnetic stir bar was added 1.0 mL of a 79.0mg (dry cell wt.)/mL suspension of Rhodococcus rhodochrous (NCIMB 11216)in 0.1 M KH₂PO₄ buffer (pH 7.5), and either 1.0 ml of 0.3 M KH₂PO₄buffer (pH 7.5) or 1.0 mL of a 10.2 mg/mL solution of polyethylenimine(BASF LUPASOL® PS, 750,000 mw) in 0.3 M KH₂PO₄ buffer (pH 7.5) at 25° C.With stirring, 2.0 mL of an aqueous solution containing glycolonitrile(1.0 M) and formaldehyde (8 mM or 0.04 mM) in distilled, deionized waterat 25° C. was added, and the reaction mixture maintained at 25° C. At 5,10, 15, and 30 minutes, a 100 μl aliquot of the reaction mixture wasremoved and mixed with 100 μl of water, 10 μl of 6.0 N HCl and 200 μl of0.25 M n-propanol in water (HPLC external standard), the mixturecentrifuged, and the resulting supernatant analyzed by HPLC to determinethe initial reaction rate and catalyst specific activity (U/g dcw)(Table 7).

TABLE 7 Dependence of Rhodococcus rhodochrous (NCIMB 11216) nitrilasespecific activity on concentration of formaldehyde with and withoutadded polyethylenimine (PEI). glycolonitrile formaldehyde PEI specificactivity (mM) (mM) (mg/mL) (U/g dcw) 500 4.0 0 37.9 500 4.0 5.0 45.4

1: A process for improving the specific activity of an enzyme catalystwhen enzymatically converting glycolonitrile to glycolic acid, saidprocess comprising: (a) providing a set of reaction componentscomprising: (i) an aqueous solution of glycolonitrile comprising atleast 0.01 ppm formaldehyde; (ii) an enzyme catalyst comprising apolypeptide having nitrilase activity wherein said polypeptide has theamino acid sequence set forth in SEQ ID NO:4; wherein said enzymecatalyst comprises a specific activity for hydrolyzing glycolonitrile toglycolic acid; and (iii) an effective amount of at least one amineprotectant selected from the group consisting of: a) a compound of theformula[R₃]_((1+X))—NH_((2−X))  wherein X is 0 or 1 and R₃ is independently C1to C20 hydrocarbyl group or substituted hydrocarbyl group, wherein R₃optionally comprises one or more ether linkages; with the proviso that(i) the substituted group is preferably not a cyano group capable ofreacting with the enzyme catalyst and (ii) R₃ is not a carbonyl group,b) a polyamine polymer comprising an effective number of free aminegroups; and c) an amine-functionalized material comprising an effectivenumber of free amine groups; wherein the amine protectant is notnaturally-produced by said enzyme catalyst; (b) combining the set ofreaction components under suitable aqueous reaction conditions wherebyglycolic acid is produced; wherein said specific activity is increasedby the addition of said at least one amine protectant; and (c)recovering the glycolic acid or salt thereof produced in (b). 2.(canceled) 3: The process of claim 1 wherein the amine protectant issoluble under said suitable aqueous reaction conditions. 4: The processof claim 1 wherein the amine protectant is insoluble under said suitableaqueous reaction conditions. 5: The process of claim 4 wherein theenzyme catalyst is not immobilized in a matrix comprising said amineprotectant. 6: The process of claim 4 wherein the enzyme catalyst isimmobilized in an insoluble matrix comprising said amine protectant. 7:The process of claim 6 wherein the enzyme catalyst is immobilized in oron an insoluble matrix comprising polyethylenimine. 8: The process ofclaim 7 wherein the enzyme catalyst is immobilized in or on aglutaraldehyde-polyethyleneimine crosslinked carrageenan particle orbead. 9: The process of claim 1 wherein the polyamine polymer isselected from the group consisting of2-amino-2-deoxy-(1→4)-β-D-glucopyranan, polyalkyleneamine polymershaving alkylene moieties of 2 to 12 carbon atoms, polyethylenimine,polyallylamine, polyvinyl alcohol/polyvinylamine copolymers,D-polylysine, L-polylysine, mixtures of D/L polylysine, polyethyleniminecross-linked with glutaraldehyde, and mixtures thereof. 10: The processof claim 9 wherein the polyamine polymer has an average molecule weightranging from 1,000 Daltons to 2,000,000 Daltons. 11: The process ofclaim 1 wherein the amine-functionalized material is selected from thegroup consisting of an amine-functionalized polymer and anamine-functionalized support material. 12: The process of claim 11wherein the amine-functionalized polymer is selected from the groupconsisting of amine-functionalized polysaccharides, amine-functionalizedglycans, amine-functionalized agarose, amine-functionalized carrageenan,amine-functionalized alginate, amine-functionalized dextran,amine-functionalized cellulose, amine-functionalized methacrylates,amine-functionalized polyurethanes, amine-functionalized polyesters,amine-functionalized nylons, amine-functionalized polystyrene, andamine-functionalized polyvinyl alcohols. 13: The process of claim 11wherein the amine-functionalized support material is selected from thegroup consisting of amine-functionalized alumina, amine-functionalizedsilica, amine-functionalized magnetite, amine-functionalized controlledpore glass, weakly basic anion exchange resins comprising one or moreprimary or secondary amine groups, aminopropylsilated glass beads,ω-aminohexylagarose, ω-aminododecylagarose, and ω-aminoethylagarose. 14:The process of claim 13 wherein the amine-functionalized supportmaterial is functionalized with polyethylenimine. 15: The process ofclaim 14 wherein the amine-functionalized support material is selectedfrom the group consisting of polyethyleneimine-functionalized silica andpolyethyleneimine-functionalized polystyrene. 16: The process of claim 1wherein the polypeptide having nitrilase activity is derived from a hostcell from a genus selected from the group consisting of Acidovorax,Rhodococcus, Nocardia, Bacillus, and Alcaligenes. 17: The process ofclaim 1 wherein the enzyme catalyst is in the form of whole microbialcells, permeabilized microbial cells, one or more cell components of amicrobial cell extract, partially purified enzyme, or purified enzyme.18: The process of claim 17 wherein the enzyme catalyst is a transformedmicrobial host cell selected from the group consisting of Comamonas sp.,Corynebacterium sp., Brevibacterium sp., Rhodococcus sp., Azotobactersp., Citrobacter sp., Enterobacter sp., Clostridium sp., Klebsiella sp.,Salmonella sp., Lactobacillus sp., Aspergillus sp., Saccharomyces sp.,Yarrowia sp., Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp.,Candida sp., Hansenula sp., Dunaliella sp. Debaryomyces sp., Mucor sp.,Torulopsis sp., Methylobacteria sp., Bacillus sp., Escherichia sp.,Pseudomonas sp., Rhizobium sp., and Streptomyces sp. 19: The process ofclaim 18 wherein the transformed microbial host cell is selected fromthe group consisting of Bacillus sp., Pseudomonas sp., and Escherichiasp. 20: The process of claim 19 wherein the transformed microbial hostcell is Escherichia coli. 21-25. (canceled)